Novel acyl-dipeptide-like compounds, a method for preparing the same and pharmaceutical compositions containing such products

ABSTRACT

The invention relates to the field of chemistry and more specifically to the field of medicinal chemistry. 
 
The invention is directed to N-acyl-dipeptide-like compounds having the general formula I  
                 
         wherein substituents A, B, X, Y, R 1 , R 2 , and subscripts n, m, p and q have the same meanings as those given in the claims. The invention is equally directed to pharmaceutical compositions containing as an active ingredient at least one compound of general formula I either in acid or salt form with an organic or mineral base. The compounds persuant to the invention display interesting pharmacological properties which make them useful as drugs.

FIELD OF THE INVENTION

The present invention relates to the field of chemistry and morespecifically to the field of medicinal chemistry

More particularly, it is directed to dipeptide-like compounds derivedfrom hydroxylated amino acids, the free amine functional groups of whichare subject to amide formation by means of fatty acids.

The invention is specifically concerned with N-acyl-dipeptide-likecompounds at least one hydroxyl group of which is esterified by an acidgroup in the neutral or charged form, having the general formula I

wherein R₁ and R₂ each designate an acyl group derived from a saturatedor unsaturated straight or branched chain-carboxylic acid having from 2to 24 carbon atoms, which is unsubstituted or bears one or moresubstituents selected among hydroxyl, alkyl, alkoxy, acyloxy, amino,acylamino, acylthio and ((C₁₋₂₄)alkyl)thio groups,

subscripts m, p and q are integers ranging from 1 to 10,

subscript n is an integer ranging from 0 to 10,

X and Y each designate a hydrogen or an acid group either in neutral orcharged form,

provided that at least one of substituents X and Y designates an acidgroup in neutral or charged form,

A and B designate, independantly from each other, an oxygen atom, asulfur atom or an imino group —NH—.

Acid groups X and Y are preferably selected among the following groups:

carboxy [(C₁₋₅)alkyl]

CH—[(CH₂)_(m)COOH][(CH₂)_(n)COOH] with m=0 to 5 and n=0 to 5

phosphono [(C₁₋₅)alkyl]

dihydroxyphosphoryloxy[C₁₋₅)alkyl]

dimethoxyphosphoryl

phosphono

hydroxysulfonyl

hydroxysulfonyl[(C₁₋₅)alkyl]

hydroxysulfonyloxy [(C₁₋₅)alkyl]

Where substituents X and/or Y designate an acid group in neutral form,reference is made to the free carboxylic, sulfonic or phosphoric form.Where the acid group is in charged form, reference is made to thecarboxylic, sulfonic or phosphoric salt form, namely by addition of anorganic or mineral base, preferably one intended for therapeutic use. Incase where bases are not intended for therapeutic use, such basesprovide a means for easy identification, purification and separation.

Similar considerations apply where X and/or Y designate a carboxylalkyl,alcenylbiscarboxylic, hydroxysulfonyl, hydroxysulfonylalkyl,hydroxysulfonyloxyalkyl, phosphonoalkyl, phosphoryloxyakyl group.

Salt forming bases intended for therapeutic use mainly include alkalinebases such as sodium, potassium or lithium hydroxides, ammonium salts,alkali earth metal bases such as calcium or strontium hydroxide,magnesium salts, ferrous metal salts and the like, organic bases such asthose derived from primary, secondary, tertiary amines such asmethylamine, diethylamine, monoethanolamine, diethanolamine,benzylamine, N-methylbenzylamine, veratrylamine, trimethoxybenzylamine,basic amino acids such as lysine and ornithine or amino sugars.

Examples of bases not intended for therapeutic use are brucine,strychnine, agmatine, homarine, glucosamine, N-methylglucosamine orN-methylmorpholin. As previously stated, salts derived therefrom willserve as separation and identification means.

When m is equal to 1 and n is equal to 0, the molecule of interestderives from serine. Where m is equal to 2 and n is equal to 0, themolecule being considered derives from homoserine. If m is equal to 3and n is equal to 0, reference is made to a pentahomoserine compound. Ifm is equal to 4 and n is equal to 0, reference is made to ahexahomoserine compound.

Where p is equal to 3 and q is equal to 1, the product of interest maybe a citrulline, ornithine or arginine compound. Where p is equal to 4and q is equal to 1, reference is made to a homoarginine or lysinecompound.

Among dipeptide-like compounds which are herein included, specialattention is devoted to compounds of general formula I which arecurrently preferred:

wherein R₁ and R₂ each designate an acyl group derived from a saturatedor unsaturated, straight or branched chain-carboxylic acid having from 2to 24 carbon atoms, which is unsubstituted or bears one or moresubstituents selected from the group comprised of hydroxyl, alkyl,alkoxy, acyloxy, amino, acylamino, acylthio and ((C₁₋₂₄)alkyl)thiogroups,

subscripts m, p and q are integers ranging from 1 to 10,

subscript n is an integer ranging from 0 to 10,

X and Y each designate a hydrogen atom or a phosphono group.

and namely

-   3-(3-dodecanoyloxytetradecanoylamino)    9-(3-hydroxytetradecanoylamino)4-oxo-5-azadecan-1,10-diol 1 and/or    10-dihydrogenphosphate and its addition salts formed with an organic    or a mineral base,-   3-(3-dodecanoyloxy-tetradecanoylamino)    9-(3-hydroxytetradecanoylamino)4-oxo-5-azadecan-1,10-diol    1,10-bis(dihydrogenphosphate) and its addition salts formed with an    organic or a mineral base,-   3-(3-hydroxytetradecanoylamino)    9-(3-dodecanoyloxytetradecanoylamino)4-oxo-5-azadecan-1,10-diol    1,10-bis(dihydrogenphosphate) and its addition salts formed with an    organic or a mineral base,-   3-(3-dodecanoyloxytetradecanoylamino)    9-(3-hydroxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol    1-dihydrogenophosphate and its addition salts with an organic or    mineral base,-   3-(3-hydroxytetradecanoylamino)    9-(3-dodecanoyloxytetradecanoylamino)4-oxo-5-azadecan-1,10-diol    1-dihydrogenphosphate and its addition salts formed with an organic    or a mineral base-   3-(3-hydroxytetradecanoylamino)    9-(3-dodecanoyloxytetradecanoylamino)4-oxo-5-azadecane-1,10-diol    10-dihydrogenphosphate and its addition salts formed with an organic    or a mineral base.

R₁ and R₂ are meant to include saturated or unsaturated, branched orstraight chain-acyl derivatives having a variable size chain, ofdistinct or identical nature, which can bear one or more substituentsselected from the group comprised of alkyl, amino, acylamino, hydroxyl,alkoxy, acyloxy acylthio and alkylthio groups,

Examples of such acylated, substituted derivatives are ricinoleyl,12-hydroxystearoyl, 2-hydroxy-3-methylbutyroyl,3-hydroxy-2-aminopentanoyl, palmitoyl, elaidyl, eleostearoyl,arachidoyl, arachidonyl, gadoleyl, behenyl, erucyl, 8-methyldecanoyl,9-methyldecanoyl, docosohexaenoyl or eicosapentaneoyl radicals.

Compounds of general formula I and notably mono- and bis-phosphorylatedcompounds referred to in code names as OM-294-MP (MP) and OM-294-DP(DP), respectively, have distinctive interesting pharmacologicalproperties, mainly with regard to immunomodulation. They areparticularly relevant in the treatment of diseases related to adeficiency in the immune defense system or an overexpression of immuneresponses, depending on doses being used. They find equally use incancer therapy and as adjuvants or response enhancers in formulatingvaccines.

Other applications include use as vectors for molecules of therapeuticinterest due to their ability to form non covalent complexes based onhydrophilic or hydrophobic interactions. Their amphophilic characterenhances formulation and transport of molecules of therapeutic interestto the membrane receptors, as well as the cell membranes and cytoplasm.They can be used alone or in conjunction with a molecule of therapeuticinterest by administering them through oral, parenteral, rectal,topical, subcutaneous or submucosal route. They can be used solely or incombination with a molecule of therapeutic interest by carrying outextemporaneous incubation ex vivo with blood cells in order to promoteformation of immunocompetent cells before injecting them back in vivousing parenteral administration.

MP and DP molecules display similar properties, as adjuvants for theimmune system when used for example in vaccination, in combination withthe appropriate antigens, against diseases of viral, parasitic,microbial or fungal origin. In contrast, the compounds according to theinvention show utterly different properties regarding their capacity toinduce cytokine production or maturation of immunocompetent stem cellsderived from hematopoietic and lymphoid organs.

MP compound promotes maturation and differenciation of monocytes intofunctional dendritic cells, in presence or absence of the appropriateantigen and acts in promoting humoral and cell mediated immunity. DPcompound shows, on the other hand, antitumoral properties.

The compounds in accordance with the invention are particularlyinteresting due to their low toxicity. They are used for treating humansand animals in doses ranging from 0.025 mg to 100 mg per unit dosage andfrom 0.05 to 200 mg daily.

The present invention is equally directed to a method for obtainingdipeptide-like compounds of general formula I, which comprises the stepsof blocking amine functional groups in positions (q+1) and ω of diaminoacid by blocking reagents which readily undergo acidolysis andhydrogenolysis, respectively, reacting the still free carboxylicfunctional group with a reducing agent to yield a corresponding alcohol,freeing the amine functional group in position (q+1) and then acylatingby means of a carboxylic acid functional deirvative of formula R₂OH,wherein R₂ is as defined above, and subsequently freeing the terminalamine functional group by hydrogenolysis to yield the diamino alcohol ofgeneral formula II

wherein R₂ designates an acyl group derived from a saturated orunsaturated, straight or branched chain-carboxylic acid having from 2 to24 carbon atoms, which is unsubstituted or bears one or moresubstituents as defined above,

p and q each designate an integer ranging from 1 to 10,

which amino alcohol is condensed in presence of a peptide condensingagent in an inert solvant, together with a ω-hydroxy, ω-amino or ω-thioamino acid compound of general formula III

wherein R₁ designates an acyl group derived from a saturated orunsaturated, straight or branched chain-carboxylic acid having from 2 to24 carbon atoms, which is unsubstituted or bears one or moresubstituents as defined above

m is an integer ranging from 1 to 10,

and n is an integer ranging from 0 to 10,

and X is an acid group as specified previously which is optionally in anester form

in order to produce a dipeptide-like compound of general formula IV

wherein substituents R₁, R₂, and subscripts n, m, p and q have the samemeanings as specified above,

the terminal free alcohol functional group of which can be—ifnecessary—alkyl or acyl or otherwise substituted by an alkyl or acyl oran otherwise substitution reagent, if needed, in presence of a couplingagent, and subjected to a catalytic hydrogenation or some otherdeprotection treatment in order to obtain the derivative of generalformula I

wherein A, B, as well as substituents and subscripts X, Y, R₁, R₂, n, m,p and q have the same meanings as those given above.

The invention is also directed to a method for obtainingphosphodipeptide-like compounds of general formula I′

wherein R₁ and R₂ each designate an acyl group derived from a saturatedor unsaturated, straight or branched chain-carboxylic acid having from 2to 24 carbon atoms, which is unsubstituted or bears one or moresubstituents selected from the group comprises of hydroxyl, alkyl,alkoxy, acyloxy, amino, acylamino, acylthio and ((C₁₋₂₄)alkyl)thiogroups,

subscripts m, p and q are integers ranging from 1 to 10,

subscript n is an integer ranging from 0 to 10,

X and Y each designate a hydrogen atom or a phosphono group, whichconsists in blocking amine functional groups in positions (q+1) and ω ofa diamino acid of formula H₂N(CH₂)_(p)CHNH₂(CH₂)_(q+1)COOH by blockingreagents which readily undergo acidolysis and hydrogenolysis,respectively, reacting the still free carboxylic functional group with areducing agent to yield a corresponding alcohol, freeing the aminefunctional group in position (q+1) and then acylating by means of acarboxylic acid functional deirvative of formula R₂OH wherein R₂ is asdefined above, then freeing the terminal amine functional group byhydrogenolysis to obtain an amino alcohol of general formula II

wherein R₂ designates an acyl group derived from a saturated orunsaturated, straight or branched chain-carboxylic acid having from 2 to24 carbon atoms, which is unsubstituted or bears one or moresubstituents as specified above,

p and q designate an integer ranging from 1 to 10

which amino alcohol is condensed in presence of a peptide condensingagent in an inert solvant, together with an ω-hydroxy amino acidfunctional derivative of general formula III′:

wherein R₁ is an acyl group derived from a saturated or unsaturated,straight or branched chain-carboxylic acid having from 2 to 24 carbonatoms, which is unsubstituted or bears one or more substituents,

m is an integer ranging from 1 to 10,

n is an integer ranging from 0 to 10,

and X is dialkyloxy- or diaryloxy-phosphoryl radical of formula

to yield the peptide-like compound of general formula IV′

wherein substituents R₁, R₂, and subscripts m, n, p and q are as definedabove, and R is a radical which readily undergoes hydrogenolysis, theother alcohol functional group of which can be—if desired—phosphorylatedby a phosphorylating agent in presence of a coupling agent, if needed,and subjected to a catalytic hydrogenation on one hand in order tounblock the alcohol functional group optionally present on acyl group R₂and on the other, free the phosphate functional group and then unblockthrough hydrogenolysis the second optionnally present phosphatefunctional group, in order to obtain the derivative of general formula V

wherein Y designates either a hydrogen atom or a phospono group, andoptionally performing the further step of salt formation by means of anorganic or mineral base.

Stereochemistry of chiral centers of acylamino groups is determined byinitially used amino acid configuration whereas stereochemistry ofacylamino groups depends on initially used fatty acid configuration. Onecan start from a diamino acid having L or D configuration or of aracemic nature. One can start from a hydroxylated amino acid of L, Dconfiguration or of a racemic mixture. All such stereoisomers ordiastereoisomers are included in the scope of the invention.

The method according to the invention can still further be defined bythe following currently preferred operating procedures which areoutlined is reaction schemes 1, 2 and 3 (FIGS. 33, 34 and 35):

1. Blocking the amine functional group in position ω of the ornithinederivative chain is accomplished by N-benzyloxycarbonyl substitutionafter initially reacting the acid functional group with a copper salt,in alkaline medium, reacting this copper carboxylate with benzylchloroformate and freeing the carboxylic functional group by chelatingcopper in an acid environment, in order to obtain anN-benzyloxycarbonyl-substituted derivative, according to the methoddisclosed in “Organic Preparations and Procedures International, 23(1992): 191-194”.

2. Blocking the amine functional group in position a of the ornithinederivative carboxyl moiety is performed by terbutyloxycarbonylsubstitution by means of an alkyl pyrocarbonate such as terbutylpyrocarbonate in alkaline medium.

Terbutyl pyrocarbonate reacts with the proximal amine functional groupto give the ω-benzyloxycarbonylamino α-terbutylcarbonylamino carboxylicderivative.

3. Conversion of the carboxylic functional group into a primary alcoholfunctional group is effected according to the method disclosed inTetrahedron Letters, 32 (1991) 923-926 which consists in reacting thecarboxylic derivative with an alkyl chloroformate, such as isobutylchloroformate, to form a mixed anhydride which is reduced by means of analkaline or an alkali-earth metal borohydride, to finally yield thecorresponding hydroxylated derivative, having a primary alcoholfunctional group.

4. Removal of the terbutyloxycarbonyl group in position α is performedusing trifluoroacetic acid which at the same time allows the formationof an amine functional group corresponding trifluoroacetate.

5. Acylation of the thus freed amine functional group is accomplishedstarting from a trifluoroacetic salt by means of a mixed anhydrideprepared from R₂OH acid and an alkyl chloroformate.

6. Freeing of the terminal amine functional group is accomplished byhydrogenolysis in presence of a noble metal-based catalyst such asplatinium, palladium on a carbon or iridium support material.

7. Peptide coupling or linkage between the amino compound of formula IIand the phosphoryl derivative of formula III′ is accomplished inpresence of a coupling agent such as1-isobutyloxy-2-isobutyloxycarbonyl-1,2-dihydroquinoline in an inertsolvent such as a halogen-containing solvent, or in presence of acarbodiimide.

Hence, there is obtained a dipeptide-like compound of general formula(IV′) the hydroxyl functional group of which optionally born by the acylgroup R₂ is blocked.

8. Freeing of the hydroxyl functional group of the acyl group R₂involves hydrogenolysis in presence of a noble metal such as palladium,applied on a substrate like carbon.

9. Freeing of the phosphoric group is accomplished by catalytichydrogenation in presence of a noble metal oxide such as platiniumoxide.

10. Phosphorylation of the dipeptide-like derivative IV′ is accomplishedin a two-step process (Helv. Chim. Acta, 70 (1987), 175) During thefirst step, compound IV′ is reacted with a dialkyl or diaryl-N,N-dialkylphosphoramidite, in presence of a coupling agent such as [1H]-tetrazolein a polar solvent such as tetrahydrofurane; the phosphite thus formedis then oxidized into a phosphate by means of an aromaticperoxycarboxylic acid such as for instance peroxyphtallic acid,m-chloroperbenzoic acid or nitroperbenzoic acid. Freeing of thephoshoric group Y (formula V) is done by catalytic hydrogenation inpresence of a noble metal such as palladium impregnated on carbon.

11. Phosphorylation of the homoserine derivative is effected by means ofa diphenylphosphoryl halide in presence of pyridine andN,N-dialkylaminopyridine (Helv. Chim. Acta, 58: (1975), 518), afterblocking the amine functional group by terbutoxycarbonyl substitution bymeans of terbutyl pyrocarbonate in alkaline medium and blocking thecarboxylic functional group following the formation of a cesium salt,and benzylation by means of a benzyl halide in dimethylformamide ordimethylacetamide.

12. Acylating the nitrogen atom of the homoserine derivative isaccomplished by deprotecting the amine functional group bytrifluoroacetic acid to obtain the amine trifluoroacetic salt, andreacting with the mixed anhydride resulting from the reaction betweenthe carboxylic acid R₁OH and an alkyl chloroformate in presence of areactive amine such as N-methylmorpholin.

The invention further relates to intermediates of general formula II andgeneral formulae III and III′, either in the form of a pure enantiomeror a mixture of stereoisomers.

The invention still relates to pharmaceutical compositions containing asan active ingredient at least one compound of general formula I, eitherin neutral or charged form, in combination or in admixture with a nontoxic, pharmaceutically acceptable, inert excipient or carrier.

The invention relates more specifically to pharmaceutical compositionscontaining as an active ingredient at least one salt of a compound ofgeneral formula I, together with an organic or mineral base intended fortherapeutic use.

The invention still further relates to pharmaceutical compositions basedon a compound of general formula I, either in the form of a pureenantiomer or in the form of a mixture of stereoisomers, in combinationor in admixture with a pharmaceutical excipient or carrier.

Among pharmaceutical formulations herein contemplated, mention should bemade of those which are suitable to administration by mucosal,transcutaneous, topical, parenteral, digestive route or inhalation suchas for instance coated or uncoated tablets, capsules, injection solutesor suspensions, spray, gels, plasters or rapid absorption solutes.

In preference, the compounds of the invention are administered byinjection as aqueous solutions or suspensions, optionally neutralized byan amine or a hydroxyalkylamine.

The following non limiting examples further illustrate the invention.They are outlined in reaction schemes 1 to 6 (FIGS. 33-38).

EXAMPLE I4-(diphenyloxyphosphoryloxy)-2-[(R)3-dodecanoyloxytetradecanoylamino]butanoicacid 1. Nα-Terbutyloxycarbonyl-DL-homoserine

2 g of homoserine (16.78 mmol) were dissolved in 20 ml of water and tothe solution 16.78 ml of 1 M NaOH and 3.006 g of cesium carbonate (9.23mmol) were added. After stirring for 5 minutes, the solution was cooledin an ice/water bath. 60 ml of dioxane and terbutyl pyrocarbonate werethen added. The reaction mixture was kept under stirring in an ice-coldwater bath for 1 hour and thereafter at room temperature for 5 hours.The solvent was subsequently removed under vacuum. The dry residue wasdirectly used in the next step.

2. Nα-Terbutyloxycarbonyl-benzyl-DL-homoserinate

To the residue of step 1, 20 ml of dimethylformamide were added and thesolvent was evaporated to dryness then to the reaction mixture wereadded 60 ml of dimethylformamide and 4.5 ml of benzyl bromide (20.13mmol). At this point, a white precipitate formed. The mixture was keptunder stirring for 16 hours. The solvent was then driven away undervacuum. The residue was depleted or extracted twice with 20 ml of ethylacetate. The organic layer was respectively washed with water (20 ml)and with brine (20 ml), then dried on anhydrous magnesium sulfate. Thesolvent was evaporated and the residue was used as such in the nextstep.

3. benzylNα-Terbutyloxycarbonyl-O-(diphenyloxyphosphoryl)-DL-homoserinate

The residue of the previous step was dried under high vacuum thendissolved in methylene chloride (60 ml). 4.11 g of4-dimethylaminopyridine (33.56 mmol) were then added to the solution,the reaction mixture was stirred for 10 minutes, and 12 ml of pyridineand 6.95 ml of chlorophosphate (33.56 mmol) were then added. Thesolution was stirred at room temperature for 18 hours then washed with 1N hydrochloric acid (5×20 ml), water (30 ml) and brine (30 ml). Theorganic layer was dried over anhydrous magnesium sulfate and the solventwas driven away under vacuum. The residue was purified by flashchromatography (hexane/ethyl acetate=4:1). The main fraction wasconcentrated to cristallize the residue. As a result, there was obtained7.49 g of phosphorylated product, that is a yield of 82.4%. Meltingpoint: 63.5-64.0° C.

4. Benzyl O-(diphenyloxyphosphoryl)-DL-homoserinate

The phosphorylated product of the previous step (7.88 g i.e 15.4 mmol)was dissolved in 15 ml of trifluoroacetic acid and the solution was keptunder stirring at room temperature for 2.5 hours. The solvent was thendriven away under vacuum, the residue was purified by flashchromatrography (MeOH/CH₂Cl₂=10:1). The main fraction was concentratedand the residue was cristallized at room temperature. As a result, 7.17g of phosphorylated product were recovered (88.9% yield). This was usedin the next step with no further work-up.

5. Benzyl2-[(R)-3-Dodecanoyloxytetradecanoylamino]-4-(diphenyloxyphosphoryloxy)butanoate

4.284 g (10.07 mmol) of (R) 3-dodecanoyloxytetradecanoic acid preparedacccording to the method disclosed in Bull. Chem. Soc. Jpn., 60 (1987),2205-2214, were dissolved in 30 ml of tetrahydrofurane and the solutionwas cooled down to −15° C. in an ice-cold brine bath. 1,108 ml (10.07mmol) of N-methylmorpholin and 1.31 ml (10.07 mmol) of isobutylchloroformate were then added. Stirring was continued for 30 minutes. Tothe reaction mixture, there was added 5.724 g (10.07 mmol) of benzyl0-(diphenyloxyphosphoryl)-DL-homoserinate in a mixture of 30 ml oftetrahydrofurane and 5 ml of triethylamine. After stirring overnight atroom temperature, the solvent was driven away under vacuum and 20 ml ofwater were added to the residue. The mixture was then extracted withethyl acetate (2×30 ml). The organic layers were pooled, washed insuccession with water (20 ml) and brine (20 ml) and dried over magnesiumsulfate. The solvent was evaporated and the residue was purified byflash chromatography (hexane-ethyl acetate 2:1, R_(f)=0.29); yield 7.455g i.e 87.1% m.p. 31.0°-32.1° C., ¹H-NMR (CDCl₃, 250 MHz), δ in ppm:7.4-7.1 (m, 15H), 6.90 (2d, 1H, ³J=7.6 Hz, NH), 5.3-5.1 (m, 3H), 4.7 (m,1H), 4.35 (m, 2H), 2.45 (m, 2H), 2.4-2.1 (m, 4H), 1.6 (m, 4H), 1.4-1.1(m, 34H), 0.9 (t, 6H). ¹³C-NMR (CDCl₃, 63 MHz), δ in ppm: 173.01,171.08, 169.66, 150.18, (d, ²J_(P,C)=7.1 Hz), 135.01, 129.60, 128.33,128.14, 127.96, 125.21, 119.80 (d, ³J_(P,C)=5.0 Hz), 70.69, 67.05, 65.19(d, ²J_(P,C)=5.6 Hz), 49.13, 40.97, 40.77 (2 diast.), 34.20, 33.98,33.82, 31.70, 29.42, 29.34, 29.14, 28.94, 25.01, 24.47, 13.91.

6.4-(diphenyloxyphosphoryloxy)-2-[(R)-3-dodecanoyloxytetradecanoylamino]-butanoicacid

A solution was prepared from the benzyl ester obtained in step 5 (2.23 gi.e. 2.6 mmol) in 300 ml of HPLC-grade methanol in a three neck-roundflask and then 1.0 g of carbon-10% palladium was added. Air contained inthe round flask was discharged under vacuum, and the flask was loadedwith hydrogen gas under atmospheric pressure.

The reaction mixture was stirred at room temperature for 1 hour, thecatalyst was then quickly filtered off on a membrane and the filtratewas concentrated to obtain a colorless liquor. This product washomogeneous as assessed by thin layer chromatography and NMR, and wasused directly with no further purification treatment in the couplingstep; Rf=0.75 (dichloromethane-methanol-triethylamine, 10:1:0.5). ¹H-RMN(CDCl₃, 250 MHz), δ in ppm: 7.4-7.1 (m, 10H), 6.85 (2d, 1H, NH), 5.15(m, 1H), 4.6 (m, 1H), 4.35 (m, 2H), 2.45 (m, 2H), 2.4-2.15 (m, 4H), 1.6(m, 4H), 1.4-1.1 (m, 34H), 0.9 (t, 6H). ¹³C-NMR (CDCl₃, 63 MHz), δ inppm: 173.35, 171.30 (2 diast.), 172.75, 170.37, 150.0 (d, ²J_(P,C)=7.5Hz), 129.55, 125.28, 119.71 (d, ³J_(P,C)=4.4 Hz), 70.78, 65.65, (d,²J_(P,C)=5.9 Hz), 49.00, 40.77, 40.63 (2 diast.), 34.13, 33.86, 33.76,31.59, 29.31, 29.25, 29.03, 28.82, 24.88, 24.68, 22.36, 13.76.

The4-(diphenyloxyphosphoryloxy)-2-[(R)-3-benzyloxytetradecanoylamino]-butanoicacid can be prepared using the same reaction scheme by replacing in step5 of example 1, (R)-3-dodecanoyloxytetradecanoic acid by(R)-3-benzyloxytetradecanoic acid.

EXAMPLE II(2R)-5-amino-2-[(R)-3-benzyloxytetradecanoylamino]-pentan-1-ol 1. CopperSalt of D-ornithine

To a solution of D-ornithine (5.25 g i.e. 30 mmol) in 30 ml of 1M sodiumhydroxide, 50 ml of a solution of cupric sulfate pentahydrate (3.814 gi.e. 15.3 mmol) in water were added. Stirring was continued for 2 hours.The solvent was evaporated to dryness. 60 ml of methanol were added toform a purple-colored solid which was separated, washed with dioxane andmethanol, respectively.

2. copper (2R)-5-Amino-5-benzyloxycarbonylamino)pentanoate

The purple-colored solid was dissolved in 40 ml of 1M soda lye and 70 mlof dioxane, the solution was cooled in an ice-cold water bath and 5.14ml (i.e. 36 mmol) of benzyl chloroformate were added. Stirring wascontinued in an ice-cold water bath for 3 hours and thereafter at roomtemperature for 15 hours. The purple precipitate was collected andwashed with 95% ethanol (40 ml), with water (50 ml) and with ethanol (60ml), respectively. The precipitate was dried in an oven (T<45° C., undervacuum); the yield of the two-step process was 8.27 g, i.e. 93% of thepredicted yield.

3. (2R)-5-(benzyloxycarbonylamino)-2-(terbutyloxycarbonylamino)pentanoicacid

The copper salt obtained in step 2 was dissolved in 2M hydrochloric acid(400 ml) and EDTA was added (8.15 g, 27.8 mmol) thereto. The mixture wasstirred for 2.5 hours, and neutralized to pH 7 by adding soda lye (about160 ml). A white precipitate was formed. The mixture wa stirred for 2.5hours in an ice-cold water bath. The precipitate was filtered, washedwith cold water until washing effluents were colorless, then dried in anoven under 60° C. This solid was dissolved in 156 ml of 1M NaOH and thesolution was cooled with an ice-cold water bath. To this solution, 7.7 g(35.2 mmol) of terbutyl pyrocarbonate in dioxane (160 ml) were added.The mixture was stirred at 0° C. for 45 minutes then for 16 hours atroom temperature. The organic solvent was evaporated and 70 ml of ethylacetate were added to the residue. The aqueous layer was acidified byadding 2N hydrochloric acid down to pH ≈3. The aqueous layer wasextracted once again with 100 ml of ethyl acetate. The organic layerswere combined and washed with water (30 ml) and with brine (30 ml). Thesolvent was removed under vacuum to therby provide a colorless oil afterflash chromatography purification (yield: 8.42 g in 2 steps i.e. 76.7%of the predicted yield) (Rf=0.19, dichloroethane-MeOH 20:1).

4.(2R)-5-(Benzyloxycarbonylamino)-2-(terbutyloxycarbonylamino)pentan-1ol

To a cold solution (−15° C.) of the diamino pentanoic acid derivativeobtained in step 3 (5.45 g. i.e. 14.8 mmol) in 60 ml of THF, 1.654 ml(i.e. 14.8 mmol) of N-methylmorpholin and 9.6 ml (i.e. 14.8 mmol) ofisobutyl chloroformate (IBCF) were added. The solution was stirred at−15° C. for 1 minute followed by addition of sodium borohydride (5.104 gi.e. 44.6 mmol) in 10 ml of water. The stirring was conducted at −15° C.for further 10 minutes then 400 ml of water were added to stop thereaction. The solution was extracted with ethyl acetate (100 ml×2). Theorganic layers were combined and washed with 50 ml of water and with 60ml of brine then dried over anhydrous magnesium sulfate. The solvent wasremoved and the residue recristallized from an ethyl acetate/hexanemixture (4.95 g, 94.9% yield) m.p. 47.5-48° C.

5. Unblocking of the 2,5-diaminopentan-1-ol derivative

6.32 g (18 mmol) of(2R)-5-(benzyloxycarbonylamino)-2-(terbutyloxycarbonylamino)-pentan-1-olobtained in step 4 were dissolved in 25 ml of trifluoroacetic acidfollowed by stirring the solution for 2.5 hours at room temperature. Thesolvent was then evaporated and the residue was purified by runningflash chromatrography (MeOH/CH₂Cl₂=10:1). A colorless vitreous bulkproduct was obtained as a result which melts at room temperature. Yieldwas 5.45 g in terms of trifluoroacetic salt (yield=82.7%). Thehydrochloride compound melts at 133.00-134.3° C. (recristallization frommethanol).

6.(2R)-5-(Benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetradecanoylamino]pentan-1-ol

To a previously cooled solution to −15° C., 5.27 g (15.8 mmol) of(R)-3-benzyloxytetradecanoic acid (Bull. Chem. Soc., Jpn., 60 (1987),2197-2204) in 30 ml of tetrahydrofuran, 1.89 ml (15.8 mmol) ofN-methylmorpholin and 2.21 ml of IBCF (15.8 mmol) were added. Thereaction mixture was kept under stirring at −15° C. for 30 minutes.Then, 5.25 g of trifluoroacetate salt of the preceding example (14.4mmol) in 30 ml of tetrahydrofuran and 1.44 ml of triethylamine wereadded to the solution. Stirring was continued at room temperature for 16hours then 30 ml of water and 60 ml of ethyl acetate were added; theorganic layer was separated and the aqueous layer was extracted onceagain with ethyl acetate (60 ml). The organic layers were pooled andwashed with water (30 ml) and with brine (30 ml) then dried overanhydrous magnesium sulfate. The solvent was evaporated and the residuewas recristallized from an ethyl acetate/hexane mixture (5.842 g, i.e.71.2% yield), m.p.=117.5-118° C. Rf=0.32, ethyl acetate-petroleum ether3:1. ¹H-NMR (CDCl₃, 250 MHz), 6 in ppm: 7.4-7.2 (m, 10H), 6.5 (2d, 1H,NH), 5.1 (s, 2H), 4.9 (m, 1H, NH), 4.5 (2d, AB, 2H), 3.8 (m, 2H), 3.5(m, 2H), 3.1 (m, 2H), 2.4 (m, 2H), 2.4 (m, 2H), 1.6-1.4 (m, 6H), 1.4-1.2(m, 18H), 0.9 (t, 3H). ¹³C-NMR (CDCl₃, 63 MHz), 6 in ppm: 172.24,156.49, 138.06, 136.53, 128.46, 128.04, 127.87, 76.76, 71.39, 66.60,65.44, 51.54, 41.43, 40.65, 33.76, 31.87, 29.61, 29.30, 28.01, 26.47,25.05, 22.65, 14.09.

7. (2R)-5-Amino-2-[(R)-3-benzyloxytetradecanoylamino]pentan-1-ol

In a three neck-flask, 150 mg of 20% palladium/carbon were added to thesolution of(2R)-5-(Benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetradecanoylamino]pentan-1-ol(3.0 g, i.e. 5.27 mmol) and 6 ml of triethylamine in 300 ml ofHPLC-grade ethanol. Air was discharged under vacuum then the flask wasloaded with hydrogen. The reaction mixture was stirred at roomtemperature for 2 hours then the catalyst was filtered off by membranefiltration and the filtrate was concentrated to provide a homogenouswhite solid as shown by TLC, to be used as such in the next step with nofurther purifcation, Rf=0.2, dichlormethane-methanol-triethylamine5:10.5, m.p.=47-48° C.

¹H-NMR (CDCl₃, 250 MHz), δ in ppm: 7.4-7.2 (m, 5H), 6.75 (d, 1H, NH)4.5(2d, AB, 2H), 3.9 (m, 2H), 3.5 (m, 2H), 2.3-2.6 (m, 7H), 1.7-1.2 (m,24H), 0.9 (t, 3H). ¹³C-NMR (CDCl₃, 63 MHz), δ in ppm: 171.86, 138.13,128.37, 127.87, 127.75, 76.81, 71.50, 64.57, 51.38, 41.51, 41.17, 33.89,31.82, 29.26, 28.57, 28.03, 25.07, 22.60, 14.04.

(2R)-5-amino-2-[(R)-3-dodecanoyloxytetradecanoylamino]pentan-1-ol can beobtained according to the same reaction scheme by replacing in step 6 ofexample II, (R)-3-benzyloxytetradecanoic acid by(R)-3-dodecanoyloxytetradecanoic acid.

EXAMPLE III3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decane-1,10-diol1-dihydrogenphosphate 1. Peptide Coupling

In a solution of(2RS)-4-(diphenyloxyphosphoryloxy)-2-[(R)-3-dodecanoyloxytetradecanoylamino]butanoicacid (1.0 mmol) as obtained in Example I, dissolved in 20 ml ofmethylene chloride, 363.6 mg (1.2 mmol) of IDQ(1-isobutyloxy-2-isobutyloxycarbonyl-1,2-dihydroquinoline) aresuspended. After stirring for 15 minutes, addition is made of 1.0 mmolof (2R)-5-amino-2-[(R)-benzyloxytetradecanoylamino]pentan-1-ol fromExample II, being dissolved in 10 ml of methylene chloride and thereaction mixture is kept under stirring for 4 hr.

The solution is concentrated and the residue is purified by a flashchromatography treatment (CH₂Cl₂/acetone=5:2, Rf 0.23). The solvent isremoved thus obtaining a colorless thick liquor (0.620 g i.e. 52.7%yield) of a phosphorylated dipeptide-like compound. Rf=0.49,dichloromethane-methanol-triethylamine, 10:1:0.5. ¹H-NMR (CDCl₃, 250MHz), δ in ppm: 7.40-7.15 (m, 15), 7.00 (m, 1H), 6.90 and 6.80 (2d, 2diast., 1H), 6.65 (d, 1H) (3×NH), 5.15 (m, 1H), 4.50 (m, 3H), 4.30 (m,2H), 3.85 (m, 2H), 3.45 (m, 2H), 3.15 (m, 2H), 2.41-2.14 (m, 8H),1.6-1.4 (m, 8H), 1.4-1.1 (m, 54H), 0.9 (t, 9H, 3CH₃). ¹³C-NMR (CDCl₃, 63MHz), δ in ppm: 173.11, 171.68, 170.52 (2 diast.), 169.94 (2 diast),150.0 (d, J_(PC)=7.2 Hz), 138.0 (2 diast.), 129.58, 127.99, 127.49,127.26, 125.24, 119.73 (t, J_(PC)=5.0 Hz), 76.48, 71.12, 70.71, 65.86(broad spin), 64.22, 50.96, 49.71 (broad spin), 41.46, 41.05, 39.07,34.13, 34.00, 32.70, 31.61, 29.34, 29.06, 28.87, 27.98, 25.25, 24.92,24.72, 22.38, 13.80.

2.1-(Diphenyloxyphosphoryloxy)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-10-ol

The phosphorylated dipeptide-like compound solution (488 mg i.e. 0.42mmol) obtained above and acetic acid (1.9 ml) in 65 ml of HPLC-gradeethanol were introduced in a three-neck round flask and 200 mg ofpalladium on carbon containing 10% Pd were added. Air was dischargedunder vacuum and the flask was loaded with hydrogen. The reactionmixture was stirred at room temperature for 2 hr., then the catalyst wasfiltered off by membrane filtration, the solvent was driven away undervacuum, to thereby recover the crude product with a yield of 92%. Asample of the product was purified by flash chromatography(CH₂Cl₂/acetone 5:4, Rf=0.24). As a result, a glass-like solid wasobtained. Rf=0.68, 5:2 methylene chloride-methanol. (¹³C-NMR (CDCl₃, 63MHz), δ in ppm (a few signals in doublet form due to the presence ofdiastereoisomers were observed): 173.60, 173.15, 170.67, 170.60, 170.27,170.07, 150.24 (d), 129.92, 125.66, 120.05, 119.90 (2d), 71.11, 71.05,68.83; 66.21 (broad spin), 64.71, 51.38; 50.32, 50.12, 43.25, 43.12,41.66, 41.57, 39.30, 37.26, 34.45, 32.84, 31.86, 29.62, 29.5, 29.29,29.13, 28.08, 25.57, 25.19, 24.97, 22.62, 14.03.

3.3-[(R)-3-Dodecanoyloxytetradecanoylamino-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol1-dihydrogenphosphate

In a three-neck round flask, platinium oxide (137 mg) was preactivatedwith hydrogen in absolute ethanol (5 ml) for 10 min. Addition was thenmade of a solution of1-(diphenyloxyphosphoryloxy)-3-[(R)-3-dodcanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino)decan-10-ol(411 mg i.e. 0.38 mmol) in absolute ethanol (20 ml). Air was dischargedunder high vacuum, and the flask was then loaded with hydrogen. Thereaction mixture was stirred at room temperature for 2-3 hr., thecatalyst was filtered off by membrane filtration, and finally thesolvent was driven away under vacuum. As a result, a crude product inthe form of a white solid was finally obtained. (crude product yield:98%). Rf=0.50, chloroform-methanol-water, 6:4:0.6.

3-[(R)-3-hydroxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-dodecanoyloxytetradecanoylamino]-decan-1,10-diol1-dihydrogenphosphate can be obtained starting from4-(diphenyloxyphosphoryloxy)-2-[(R)-3-benzyloxytetradecanoylamino]butanoicacid and(2R)-5-amino-2-[(R)-3-dodecanoyloxytetradecanoylamino]pentan-1-olaccording to the same reaction scheme (scheme 3) (FIG. 35).

Alternatively,3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]-decan-1,10-diol1-dihydrogenphosphate is obtained starting from aspartic acid accordingto the following reaction scheme (reaction schemes 1, 5 and 6) (FIGS.33, 37 and 38): protecting the free OH functional group of(2R)-5-(benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetradecanoylamino]pentan-1-olby a benzyloxymethyl group, freeing the 5-amino functional group of thiscompound by hydrogenolysis, effecting peptide coupling of this aminewith a monoesterified derivative of D or L-aspartic acid bearing at theamine functionality thereof either a protecting group or an(R)-3-dodecanoyloxytetradecanoyl group, freeing and reducing theterminal carboxylic functional group by means of a mixed anhydride,deprotecting, if needed, the amine functional group derived fromaspartic acid then N-acylating with an (R)-3-dodecanoyloxytetradecanoicacid derivative, phosphorylating the hydroxy functional group at C₁ andfinally unblocking the phosphate and hydroxyl functional groups throughhydrogenolysis.

EXAMPLE IV Preparation of3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol1,10-bis(dihydrogenphosphate)

1-(diphenyloxyphosphoryloxy)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-benzyloxytetradecanoylamino]decan-10-ol(985 mg i.e. 0.84 mmol) is reacted with dibenzylN,N′-diethylphosphoramidite (0.58 ml, 85% pure), in presence of[1H]-tetrazole (182 mg) in tetrahydrofurane (35 ml) for 30 minutes atroom temperature. The phosphite intermediate is oxidized by addition ofa solution of m-chloroperoxybenzoic acid (535 mg) in 25 ml of methylenechloride at a temperature range of 0° to −20° C. After 20 min., asolution of Na₂S₂O₃ (20 ml) is added to neutralize any excess oxydant,then the organic layer is diluted with ether. The organic layer isseparated, washed in succession with an aqueous solution of Na₂S₂O₃(5×20 ml), then absolution of NaHCO₃ (2×20 ml), thereafter with aqueoushydrochloric acid (20 ml), dried over MgSO₄ and concentrated. The crudeproduct is purified by flash chromatography treatment over a silica gel(CH₂Cl₂-acetone 10:3). The protected diphosphorylated derivative thusobtained (900 mg, 75% yield) (Rf 0.64, 5:2 dichloromethane-acetone) issubjected to a catalytic hydrogenation in HPLC-grade methanol (1000 ml)in presence of 10% palladium-carbon (300 mg) under atmospheric pressure,for 4 hr. at room temperature. The catalyst is filtered off by membranefiltration and the filtrate is concentrated under reduced pressure, tothereby recover crude10-(dihydroxyphosphoryloxy)-1-(diphenyloxyphosphoryloxy)-3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-[(R)-3-hydroxytetradecanoylamino]decane(Rf=0.63, chloroform-methanol-water 6:4:0.6) with a yield of 89%. Thisproduct is then submitted to a catalytic hydrogenation on platiniumoxide (380 mg) in HPLC-grade ethanol (130 ml) for 24 hr. at roomtemperature, under atmospheric pressure. The catalyst is filtered off bymembrane filtration and the filtrate is concentrated to thereby obtainthe free bis dihydrogenophosphate compound (Rf=0.20,chloroform-MeOH-water, 6:4:0.6).

3-[(R)-3-hydroxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-dodecanoyloxytetradecanoylamino]-decan-1,10-diol1,10-bis(dihydrogenphosphate) can be obtained starting from4-(diphenyloxyphosphoryloxy)-2-[(R)-benzyloxytetradecanoylamino]butanoicacid and(2R)-5-amino-2-[(R)-3-dodecanoyloxytetradecanoylamino]pentan-1-olaccording to the same reaction scheme (scheme 3) (FIG. 35).

Alternatively,3-[(R)-3-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol1,10-bis(dihydrogenphosphate) is obtained starting from aspartic acidusing the following reaction scheme (reaction schemes 1, 4 and 6):freeing the 5-amino functional group of(2R)-5-(benzyloxycarbonylamino)-2-[(R)-3-benzyloxytetradecanoylamino]pentan-1-olby hydrogenolysis, performing peptide coupling of this amine with amonoesterified derivative of D or L-aspartic acid bearing at the aminefunctionality thereof either a protecting group or a(R)-3-dodecanoyloxytetradecanoyl group, freeing and reducing theterminal carboxylic functional group by means of a mixed anhydride,deprotecting, if needed, the amine functional group derived fromaspartic acid then N-acylating with an (R)-3-dodecanoyloxytetradecanoicacid derivative, phosphorylating the hydroxy functional group at C₁ andC₁₀ and finally unblocking the phosphate and hydroxyl functional groupsthrough hydrogenolysis.

EXAMPLE V Purification and Analysis of Compounds According to theInvention

1. Purification of Monophosphorylated and Diphoshorylated Compounds

The monophosphorylated and diphosphorylated synthetic products weredissolved in a water-isopropanol mixture (1:1 vol./vol.) with 0.1%triethylamine to ajust the pH in the range of 8 to 9. The requiredamount of 2 M ammonium bicarbonate was subsequently added to achieve aconcentration of 25 mM.

The purification was run by preparative reverse phase HPLC under thefollowing conditions:

Column: Bondapack C18′ Prep Pak, 40×200 mm, 15-20 μm, 300 Å, Waters

Mobile Phase:

A: isopropanol-water (1:1, vol./vol.), 50 mM ammonium bicarbonate

B: isopropanol-water (2:8, vol./vol.), 50 mM ammonium bicarbonate

Flow rate: 40 ml/min.

Elution: Isocratic adsorption onto column: 40% B (60% A), 10 minutes.

A:B gradient: 40-80% B within 10 minutes

Isocratic elution: 80% B, 30 minutes

Washing step: 100% B, 10 minutes

Detection: UV, 210 nm (wavelength)

In the aforementioned eluting conditions, the retention time of themonophosphorylated compound varies from 25 to 30 min while that for thediphosphorylated compound varies from 18 to 25 minutes. Should thepresence of monophenyl-products be observed (incomplete deprotectiontreatment during final dephenylation), a finer purification step isrequired. This further purification is performed in the followingconditions:

Column: Kromasil C18, 21×250 mm, 5 μm, 100 Å, Macherey-Nagel

Mobile Phase:

A: isopropanol-water (1:1-v/v), 50 mM ammonium bicarbonate.

B: isopropanol-water (2:8-v/v), 50 mM ammonium bicarbonate

Flow rate: 10 ml/min.

Elution: Isocratic adsorption onto column: 40% B (60% A), 10 minutes

Isocratic Elution:

monophosphorylated compound: 80% B, 30 minutes

diphosphorylated compound: 74% B, 30 minutes

Washing step: 100% B, 10 minutes

Detection: UV; 210 and 254 nm (wavelength)

Fractions containing the monophosporylated or diphosphorylated compoundsin the form of an ammonium salt are collected and concentrated byadsorption on C18 phase Bondapack, 15-20 μm, 300 Å, Waters, The sodiumsalt of monophosphorylated or diphosphorylated compounds is obtainedthrough washing with a 10 g/l NaCl solution in water-isopropanol (9:1,v:v). After removal of excess NaCl by flowing over the column 5 volumesof a water-isopropanol mixture (9:1, v/v), the compound is eluted withpure isopropanol. This solvent is then evaporated to dryness on aRotavapor. Final dissolution is conducted with the required volume ofwater (with adjunction of 0.1% triethanolamine in case of amonophosphorylated compound) to achieve a target concentration of 2mg/ml. Sterile filtration is then performed on a 0.2 μm filter, ExpressMembrane, Millipore (if volume is less than 50 ml: the Steriflip systemis recommended, if volume greater than 50 ml: the Steritop system isrecommended).

In handling a monophosphorylated compound, it is advisable to sonicatethe solution (3×10 seconds) at room temperature before running sterilefiltration.

2. Monitoring and Yield of Purification

After termination of each step, the fractions are analyzed by reversephase analytic HPLC chromatography according to the followingconditions:

Column: Supelcosil C18, 3 μm, 4.6×150 mm, 100 Å, Supelco

Mobile Phase:

A: water:acetonitrile (1:1, v/v), 5 mM TBAP

B: water-isopropanol (1:9, v/v) 5 mM TBAP

TBAP: tetrabutylammonium phosphate

Flow rate: 1 ml/min.

Elution: A: B gradient (75: 25-0:100) within 37.5 minutes.

Detection: UV, 210 and 254 nm (wavelength)

When chromatography is conducted accordingly, the retention timesobserved for mono- and diphosphorylated compounds are 25.5±0.5 and20.8±0.5 minutes, respectively. The purification yields achieved rangefrom 57 to 94% for the monophosphorylated compound and from 71 to 92%for the diphosphorylated compound. 311 mg and 189 mg of mono- anddiphosphorylated compounds are obtained, respectively.

3. Assay and Analysis of the Final Product Purity Level

Quantitative assays and purity level analysis of the products obtainedwere conducted by HPLC/UV according to the chromatography operatingconditions stated previously. According to such assays, the puritylevels obtained for different batches of mono- and diphosphorylatedcompounds vary from 99 to 100%. To show the presence of inactiveimpurities in the UV range, LC/ES-MS analysis were conducted(electrospray type ionization, positive mode). For the latter, the (5mM) tetrabutylammonium phosphate was replaced by (25 mM) ammoniumacetate to meet the requirements of ionization at the electrosprayinterface.

Alternative determination methods were used to assay the finalsolutions. For example, quantitative analysis of total phosphates(adapted from Ames, B. N., Methods in Enzymology VII (1966), 115-117),amino acids (adapted from Hughes et al., J. Chromatography, 389: (1987),327-333) and acyl chains (adapted from Miller, L. T., Hewlett PackardApplication Note (1984), 228-237) can be listed.

4. Spectroscopic Analysis

4.1. Mass Spectrometry

ES-MS spectra (negative and positive modes) of mono- anddiphosphorylated compounds were plotted using three types of massspectrometers. (Finnigan LCQ, ion trap; Micromass Quattro II, triplestage quadrupole; Hewlett-Packard MSD, single quadrupole). ComplementaryMS/MS analysis were also conducted. Spectra demonstrating the identityand purity of said products are included in the appendix.

ES-MS Spectra (Positive Mode)

Diphosphorylated Compound:

(Micromass Quattro II: Spectrum 1; HP-MSD: Spectrum 3) (FIGS. 39 & 41)

At low energy level, a major pseudomolecular ion is observed at an m/zratio of 1014.6 [M+H]⁺. Sodium adducts at an m/z ratio of 1036.6[M+Na]⁺, 1058.6 [M-H+2Na]⁺ and at 1080.5 [M-2H+3Na]⁺ are also visible.

Depending on the degree of fragmentation, two 916.5 [M-98+H]⁺ and 818.6[M-98-H]⁺ m/z fragments are observed, a fact which demonstrates thepresence of two phosphoryl group on the molecule. As depicted byspectrum 3 (FIG. 41), the relative intensity of observed ions variesconsiderably according to the extent of energy level being applied.

Monophosphorylated Compound:

(Micromass Quattro II: Spectrum 2) (FIG. 40)

A somewhat different ionization diagram is obtained for themonophosphorylated compound due to the presence of triethanolamine(TEoA) in the solutions being analyzed. A major pseudomolecular ion isobserved at an m/z ratio of 934.4 [M+H]⁺ as well as sodium adducts[M+H]⁺ and potassium adducts [M+K]⁺ at an m/z ratio of 956.3 and 972.3,respectively. A second group of adducts at an m/z ratio of 1083.4[M+TEA+H]⁺, an m/z ratio of 1105.3 [M+TeOH+Na] and an m/z ratio of1121.3 [M+TeOH+K]⁺ is equally visible. The presence of a phosphorylgroup inside the molecule is evidenced by a fragment being detected athigh energy level corresponding to an m/z ratio of 836.4 [M-98+H]⁺.

ES-MS Spectra (Negative Mode)

Ion species observed in negative mode ES-MS spectra for mono- anddiphosphorylated compounds are quite in agreement with results obtainedin the positive mode.

FAB ionization analysis (positive mode) were also conducted. At lowresolution level, the mono- and diphosphoyrlated compounds show sodiumadducts [M+Na]⁺ at 956.5 and 1036.5 m/z ratio, respectively.

At high resolution level (3-nitro benyl alcohol matrix), a peak wasobserved at an m/z ratio of 956.667 for the the monophosphate compound,corresponding to the expected molecular formula: C₄₉H₆₉O₁₁N₃PNa(predicted mass: 956.668 amu).

For the diphosphated compound, a peak at an m/z ratio of 1036.635 wasrecorded, corresponding to the expected molecular formulaC₄₉H₉₇O₁₄N₃P₂Na (calculated mass: 1036.634 amu).

All MS analysis provided evidence of the high purity level of theobtained products.

4.2 Nuclear Magnetic Resonance

¹H-NMR and ¹³C-NMR spectra for mono- and diphosphorylated compounds weredetermined using a DPX Brucker model apparatus operating at 250.13 and62.89 MHz, respectively, and a Varian Unity Inova system operating at500-499.87 and 125.7 MHz, respectively. ³¹P-RMN spectra were recorded at121.6 mHz (DPX Brucker). Spectra showing the identity and purity of thethese products are included in the appendix.

¹H-NMR Spectra (Spectra 4 & 5) (FIGS. 42 & 43)

Monophosphorylated compound: On spectra recorded in CDCl₃+0.1%triethanolamine (TEoA) (Spectrum A), signals corresponding to threeprotons born by nitrogen atoms N(5), N(2a) and N(2b) between 7 and 9.5ppm (see magnified view of the spectral window) were observed. Signalsascribed to H—N(2a) and H—N(2b) appear in the form of 2 doublets whichshow the presence of a mixture of stereoisomers. One of thediastereoisomers is observed to be prevailing (as a result of thedifferent purification steps).

Diphosphorylated compound: On the spectrum recorded in CDCl₃—CD₃OD (3:1,v/v) (Spectrum 5), signals corresponding to H—N(5), H—N(2a) and H—N(2b)are no longer visible as a result of species exchange in presence ofCD₃OD.

Additional information regarding the assignment of differents signalswere gained from homo- and heteronuclear correlation experiments(¹H-¹H-NMR: COSY, ¹H-¹³C-NMR: HSQC & HMBC).

¹³C-NMR Spectra (Spectra 6 & 7) (FIGS. 44 & 45)

Recording of ¹³C-NMR spectra is extremely difficult to carry out due tothe rather low solubility of mono- and diphosphorylated compounds.

³¹P-NMR Spectra (Spectra 8.& 9) (FIGS. 46 & 47)

For both mono- and diphosphorylated compounds, a single peak isobserved.

EXAMPLE V Pharmacological Studies of the Compounds According to theInvention

1. Endotoxicity Determination by the Limuls Chromogenic Test

Endotoxicity was determined by a chromogenic Limuls Amoebocyte Lysatetest (Chromogenic LAL of Charles River Endosafe, batch # EK412 E,Charleston, USA). This test is based on activation by alipopolysaccharide (LPS) or structurally analogous products, of anenzymatic cascade present in LAL. This enzymatic activation isdemonstrated by cleavage of a chromogen linked to a peptide under theaction of a protease, at the final stage of this enzymatic cascadeaccording, to the following reaction scheme:

The enzymatic reaction is conducted at 37° C. and the time-coursechromogen formation is measured at 405 nm. In the final stage of thistime-course determination assay, the time required to achieve an OD of0.2 unit is recorded and the endotoxic activity is calculated based onan LPS standard (standard curve).

Results are expressed in EU (Endotoxin Unit) in relation to astandardized preparation of E. coli lipolysaccharides. For this seriesof assays, 1 EU corresponds to 0.08 mg of LPS equivalent.

The results show a relatively high degree of variability, though this isnormal for such a kind of quantitative assays which provides, inessence, an indication on magnitude. LAL testing is chiefly conducted todemonstrate the absence of pyrogens (upper limit of endotoxinconcentration) in pharmaceutical preparations. It is mandatory tocompare the quantitative assay of the pyrogen content with a given wellstandardized single series of experiments.

Results

The results (mean±standard deviation) obtained for the products of theinvention are set forth in Table (A): TABLE (a) Activation of limulusamoebocyte lysate (LAL) LAL activity in LPS equivalents Products LALactivity in EU/mg ng eq. LPS/mg OM-294-DP 56 ± 48 6.2 OM-294-MP 13 ± 2 1.4 E. coli LPS (reference) 7.7 ± 1.6 × 10⁶ 0.85 × 10⁶

The compounds of the invention are 10⁶-fold less active than LPS in theLAL test. OM-294-DP and OM-294-MP are therefore particularly interestingproducts by virtue of their low toxicity, when taken together with theirability to mediate biological activites and act as immunomodulators(both in vivo and in vitro).

2. Determination of Bone Marrow Stem Cell Proliferation of Mice inResponse to LPS Stimulation or Compounds According to the Invention

Procedure

Two six-week old male C57/BL6 mice were killed by CO₂ inhalationfollowed by cervical dislocation. The mice were washed with alcohol, andthe skin of the posterior members was entirely removed. The hip, femurand tibia bones were removed by joint disruption. The flesh was grosslyremoved using a scalpel. The bones were cleaned and the bone ends werecut with scissors. The marrrow was extracted from the bone lumen byinjecting three times 1 ml of Dulbecco's Modified Eagle Medium (DHmedium) from the the extremities which were cut with scissors. The cellswere suspended in DH medium and centrifuged at 300×g for 5 minutes, Thesupernatant fluid was discarded and the stem cells were suspended in DHmedium supplemented with 20% foetal calf serum (FCS) The cellconcentration was adjusted to 500 000 cells/ml.

Products previously dissolved in DH medium supplemented with FCS, aminoacids and antibiotics were serially diluted, directly into 96-wellmicrotiter plates. 9 dilutions are performed using a dilution factor of3.16. The products are tested in series of six and each microtiter plateincludes a negative control containing plain medium. The final volume ineach well is 100 μl. The microplates are incubated for 1 hour at 37° C.under 8% CO₂-100% RH incubator to buffer the medium. After 1 hour, 100μl of the cell suspension are added to the products and incubation iscontinued for 7 days.

Proliferation is determined by measuring the oxydation of a chromogenicsubstrate (XTT) in mitochondria of viable cells.

7 days later, the microtiter plates are centrifuged for 5 minutes at400×g, and 100 μl of the supernatant fluid are withdrawn and discarded.50 μl of a 1 mg/ml XTT sodium3-[1-phenylamino-carbonyl)-3,4-tetrazolium]-bis[(4-methoxy-6-nitro)benzenesulfonate] and 0.008 mg/ml PMS ((N-methyl dibenzopyrazine, methylsulfate) in RPMI medium are added to each well. After 8 hour incubationat 37° C. under 8% CO₂ in an incubator at 100% RH, the microtiter platesare read with a spectrophotometer at 480 nm against a standard at 690nm.

The results are expressed as mean values (+standard deviation) byplotting a dose versus response curve. The values of the negativecontrol composed of DH medium (mean±standard deviation of allexperimental data) are also graphically shown.

In this experiment, compounds according to the invention induce asignificant cell proliferation of mouse bone marrow stem cells. Theextent of such a response is nearly equal to that induced by E. coliLPS, but the minimal concentration required to induce a significantresponse is higher. The monophosphorylated product induces a moremoderate response than the diphosphorylated product. FIG. 1 depicts arepresentative experiment derived from a set of three independantstudies run on different cellular preparations.

3. Determining the Production of Nitric Oxide in the Supernatant Fluidsof Macrophage Cultures.

Procedure

Two six-week old male C57/BL6 mice were killed by CO₂ inhalationfollowed by cervical dislocation. The mice were washed with alcohol, andthe skin of the posterior members was entirely removed. The hip, thefemur and the tibia bones were removed by joint disruption. The fleshwas grossly removed using a scalpel. The bones were cleaned and the boneends were cut with scissors. The marrrow was aspirated by injectingthree times 1 ml of Dulbecco's Modified Eagle Medium (DH medium) in thebone lumen. The cells were resuspended in DH medium and centrifuged at300×g for 5 minutes. The supernatant fluid was discarded and the cellswere resuspended at a density of 40 000 cells/ml in DH mediumsupplemented with 20% horse serum (HS) and 30% L929 culture supernatant.L929 is a murine fibroblast cell line the supernatant fluid of which isrich in growth factor for macrophage (M-CSF). The cell suspension wasdivided into 12 ml aliquots in Petri dishes which were incubated for 8days in an incubator at 37° C. under 8% CO₂ and 100% RH. After 8 days,the stem cells differenciated into mature macrophage cells. Themacrophage cells were scraped off by incubating them for 45 minutes at4° C. in cold PBS buffer. After centrifugation and removal of thesupernatant fluid, the cells were resuspended in DH medium supplementedwith 5% foetal calf serum (FCS), glutamine, asparagine, arginine, folicacid, mercaptoethanol, and antibiotics (penicillin and streptomycin).The stem cells were collected and cellular density was adjusted to 700000 cells/ml.

Products previously dissolved in DH medium supplemented with FCS, aminoacids and antibiotics were serially diluted directly in 96-wellmicrotiter plates. 9 to 10 dilutions depending on the products wereconducted using a 3.16 dilution factor. The products were tested intriplicate and each microtiter plate comprised a negative controlcontaining plain medium. The final volume in each well was 100 μl. Themicrotiter plates were incubated for 1 hour in an incubator at 37° C.under 8% CO₂ and 100% RH to buffer the medium. After 1 hour, 100 μl ofthe cell suspension were added to the products and incubation wasextended for 22 hours.

After 22 hours, the microtiter plates were centrifuged, 5 minutes at400×g and 100 μl of supernatant fluid were withdrawn and transferredinto a microtiter plate. 100 μl of Griess reagent [5 mg/ml ofsulfanilamide+0.5 mg/ml of N-(1-napthtylethylene diamine) hydrochloridein 2.5% aq. phosphoric acid], were added to each well. The microtiterplates were read with a spectrophotometer at 562 nm wavelength against areference at 690 nm. The nitrite concentration was proportional tonitric oxide content. The nitrite content is determined based on astandard curve, which shows a linear relationship in the range of 1 to25 μM.

The results are expressed as mean±standard deviation after deduction ofthe negative control value and plotted as a dose versus response curve.

In this experiment, the compounds according to the invention induce theproduction of nitric oxide by murine macrophage cells in a mannerconsistent with a dose vs. response curve. The diphosphorylated productinduces proliferation to a much greater extent than E. coli LPS, but theconcentration required to induce a significant response is higher. Themonophosphorylated product induces a weaker response compared to thatobtained using the diphosphorylated product and that of E. coli LPS.FIG. 2 depicts a representative experiment derived from a set of 3independent measurements run on different cell preparations.

4. Determination of the Ability of Compounds According to the Inventionto Elicit the Production of α-TNF by Human Alveolar Macrophage Cells

Procedure

Preparation of alveolar macrophage cells: Human alveolar macrophagecells were obtained by bronchoalveolar washing (BAL) of lungs inpatients suffering from lung cancer. BAL is conducted immediately, afterpulmonary tissue surgery involving healthy parts of the pulmonary lobe.Washings are performed using 0.8% NaCl with the aid of a 50 ml capacitysyringe. Cells recovered are made up for greater than 85% of macrophagecells, the majority of other cells being lymphocytes. Aftercentrifugation, the cells are suspended into RPMI medium and the redblood cells are removed by centrifugation on (Research-Grade) FicollPack. The macrophage cells are washed 3 times with HBSS and seeded into24-well microtiter plates at a rate of 1 ml per well containing a totalof 1 000 000 cells. After incubation for 1 hour at 37° C., the resultingmacrophage cells become adherent and the wells are washed three timeswith 1 ml of HBSS in order to remove non adherent cells. After thewashing step, 1 ml of RPMI is added to each macrophage cell-containingwell.

Incubation with products and assay of α-TNF: Alveolar macrophage cellsare incubated at 37° C. under 5% CO₂ in the presence of 0.1 μg/ml, 1μg/ml and 10 μg/ml concentrations of the following products:

-   -   Negative control: RPMI    -   Positive control: E. coli LPS (serotype O5: B5, Difco, Detroit,        U.S.A.)    -   Monophosphorylated compound according to the invention        (OM-294-MP)    -   Diphosphorylated compound according to the invention (OM-294-DP)

The culture supernatants are recovered after 24 hours and analyzed for αTNF content (BioSource Cytoscreen Kit, Camarillo, Calif., U.S.A) whichhas a sensitivity of 1 pg/ml.

Results

The monophoshorylated and diphosphorylated derivatives persuant to theinvention induce moderate production of α TNF in concentration as low asfrom 10 μg/ml. The monophosphorylated derivative according to theinvention induces α TNF production to a higher extent than thediphosphorylated one. The LPS positive control induces at all threetested concentrations high production of α TNF.

The results are listed in Table (a) TABLE (a) Induction of α TNFproduction by OM-294-MP and OM-294-DP in human alveolar macrophage cellsα TNF [pg/ml] mean ± standard deviation of 3 independant experimentsProduct 0 μg/ml 0.1 μg/ml 1 μg/ml 10 μg/ml Negative control: RPMI 195 ±70 Positive control: E. coli LPS 7667 ± 115  9858 ± 2148 10390 ± 3415OM-294-MP-1 246 ± 38 353 ± 75 1049 ± 295 OM-294-MP-2 205 ± 62 291 ± 701124 ± 406 OM-294-DP-1 156 ± 66 117 ± 85  329 ± 141 OM-294-DP-2 171 ± 79 88 ± 61

5. Determining the Capacity of Compounds in Accordance with theInvention to Inhibit α TNF Production in Human Alveolar MacrophageCells, in Response to E. coli Lipopolysaccharide (LPS)

Procedure

Preparation of alveolar macrophage cells: Human alveolar macrophagecells were obtained by bronchoalveolar washing (BAL) of lungs inpatients suffering from lung cancer. BAL is conducted immediately afterpulmonary tissue surgery involving healthy portions of the pulmonarylobe. Washings are performed using 0.8% NaCl with the aid of a 50 mlcapacity syringe. Cells recovered are made up for greater than 85% ofmacrophage cells, the majority of other cells being lymphocytes. Aftercentrifugation, the cells are suspended into RPMI medium and the redblood cells are removed by centrifugation on (Research-Grade) FicollPack. The macrophage cells are washed 3 times with HBSS and seeded into24-well microtiter plates at a concentration of 1 ml per well containinga total of 1 000 000 cells. After incubation for 1 hour at 37° C., theresulting macrophage cells become adherent and the wells are washedthree times with 1 ml of HBSS in order to remove non adherent cells.After the washing step, 1 ml of RPMI is added to eachmacrophage-containing well.

Incubation with products and α TNF assay: Alveolar macrophage cells areincubated at 37° C. under 5% CO₂ in the presence of E. coli LPS (O5: B5serotype, Difco, Detroit, U.S.A.) at 1 mg/ml to which are addedsimultaneously the following products at a concentration of 10 μg/ml:

-   -   Negative control: RPMI    -   Monophosphorylated compound according to the invention        (OM-294-MP)    -   Diphosphorylated compound according to the invention (OM-294-DP)

The culture supernatant fluids are recovered after 24 hours and analyzedfor α TNF content (BioSource Cytoscreen Kit, Camarillo, Calif., U.S.A)which has a sensitivity of 1 pg/ml.

Results

The diphosphorylated derivative considerably inhibits the production ofαTNF normally induced by LPS. The monophosphorylated derivativepartially inhibits α TNF producton induced by LPS.

The results are set forth in Table (a) TABLE (a) Inhibition ofLPS-induced α-TNF production by OM-294-MP and OM-294-DP in humanalveolar macrophage cells Product α TNF [pg/ml] % inhibition RPMI(negative control) 73 — E. coli LPS (10 μg/ml) 8470  0 (positivecontrol) OM-294-MP-1 (10 μg/ml) + E. coli 4577 44 LPS (1 μg/ml)OM-294-MP-2 (10 μg/ml) + E. coli 4789 41 LPS (1 μg/ml) OM-294-DP-1 (10μg/ml) + E. coli 1267 84 LPS (1 μg/ml) OM-294-DP-2 (10 μg/ml) + E. coli1280 84 LPS (1 μg/ml)

6. Effect of OM-294-MP and OM-294-DP Products on Dendritic CellMaturation

The ability of OM-294-MP and OM-294-DP products to induce maturation ofpredendritic cells into dendritic cells was evaluated. The followingparameters were measured: FITC-Dextran conjugate incorporation andexpression of CD40, CD80, CD83, CD86 surface markers.

Procedure

Cells: Mononucleated cells of peripheral blood are isolated from buffycoats of six healthy donors. Donors did not undergo any treatment priorto blood donation.

Cell preparation: Purified monocytes by adherence selection areresuspended in RPMI-1640 medium (Sigma-Aldrich: St.-Louis, Mo., U.S.A)containing 10% of foetal calf serum, GM-CSF (10 ng/ml; IM-HGMI,Immungenex Corp., Los Angelos, Calif., U.S.A.) and IL-4 (10 ng/ml; No204-IL, R&D System, Minneapolis, Minn., USA) at a density of 1×10⁶cells/ml and divided into Petri Dishes of 10 cm in diameter (P10,Falcon, Becton Dickinson, Plymouth, UK) (10×10⁶ cells per dish P10) andcultured for 6 days (with a change to fresh medium after 3 days). Thesecells are called predendritic cells (DC-6). Maturation of predendriticcells into mature dendritic cells is achieved by incubating cells withOM-294-MP, OM-294-DP or LPS for 3 further days at concentrations setbelow under Product section. At day 9, (DC-9) cells were harvested andanalyzed for different indicators of dendritic cell maturation:assessment of CD40, CD80, CD83, CD86 surface markers as well as of theirability to take up FITC-Dextran conjugate. All theses parameters areanalyzed by an EPICS-XL-MCL model FACS (Coulter Immunology, Hialeah,Finland).

Lanzavecchia et al., J. Exp. Med., 179 (1994) 1109; Lanzavecchia et al.,J. Exp. Med., 182 (1995) 389.

Data analysis: Expression of surface markers is expressed as % of meanfluorescence of cells stimulated by LPS (positive control); FITC-Dextranconjugate take-up is calculated with respect to take-up rate for cellsmaintained in basic medium and is expressed in %. Statistic analysis byt-student test involves comparing data obtained from different testswith the data of a positive control. Data significance level is set atp<0.05.

Products: Stock solutions of OM-294-MP and OM-294-DP are prepared at aconcentration of 1 mg/ml in 0.9% NaCl/water, with 0.1% triethylaminebeing added in case of OM-294-MP. Solutions are incubated at 37° C. for20 minutes, subjected to vigorous stirring during 3 minutes then dilutedto 100 μg/ml in RPMI-1640 culture medium and used either at aconcentration of 10 mg/ml (FIGS. 3, 6, 7, 8) or at concentrationsranging from 0.02 to 25 μg/ml (FIGS. 4, 5).

Reference Product: E. coli lipopolysaccharide (LPS, DIFCO, Detroit,Mich., USA), as a 5 mg/ml stock solution in PBS. An intermediate 100μg/ml solution is prepared in RPMI 1640 culture medium. Concentrationsbeing tested are either 10 μg/ml (FIGS. 3, 6, 7, 8) or in the range of0.02 to 10 μg/ml (FIGS. 4, 5).

Results

Immature dendritic cells (DC-6) resulting from monocyte differenciation,through the joint action of GM-CSF and IL-4, are able to incorporateFITC-Dextran conjugate. During the maturation process, cells lose theirability to incorporate the FITC-Dextran conjugate. Analysis areconducted upon reaching the DC-9 differenciation stage.

Results are expressed in terms of % incorporation of FITC-Dextranconjugate observed in non stimulated cells (basic medium) FIG. (3).Cells treated with LPS or OM-294-MP retain respectively only 10% and 19%of their phagocytic capacity, whereas cells stimulated with OM-294-DPtotally retain their ability to incorporate FITC-Dextran conjugate (98and 99%). A dose vs. response curve indicates that OM-294-MP has anoutstanding capacity to induce differenciation of DC-6 into DC-9 cellsat concentrations ranging from 0.02% g to 25 μg per ml, see FIG. (4)where low concentrations and FIG. (5) where higher concentrations havebeen tested.

Expression of co-stimulating surface markers is another criterion toassess DC maturation. Expression of CD40, CD80, CD83, CD86 is tested.Results are expressed in terms of % of mean fluorescence based onLPS-induced expression of these markers.

OM-294-MP increases the expression of all surface markers being tested:CD40 (39%), CD80 (62%), CD83 (60%), CD86 (77%) see FIGS. (6, 7, 8, 9).

OM-294-DP exerts an effect similar to that of basic culture medium uponexpression of the investigated markers. Such an effect does not exceed20% of LPS action.

7. Effect of OM-294-MP and OM-294-DP Products on Production of α TNF andIL-12 p70 by Monocytes and Predendritic Cells at DC-6 Stage

DC-6 cells (5×10⁵/500 μl of medium) are stimulated during 4 hr., 6 hr.,and 24 hr., either by LPS (10 μg/ml) or by OM-294-MP (10 μg/ml) orOM-294-DP (10 μg/ml).

Procedure

In vivo experimental conditions: Mononuclear cells of peripheral bloodare recovered from buffy coats of 6 healthy donors (donors did notundergo any treatment prior to blood donation). Monocytes are isolatedin a Ficoll gradient then purified by adherence selection. Looselyadherent monocytes are harvested and one fraction of the cells is storedas monocytes. Purified monocytes are resuspended into RPMI-1640 mediumcontaining 10% of FCS, at a rate of 1×10⁶ cells/ml and divided intoPetri dishes measuring 10 cm in diameter (P10-Falcon, Becton Dickinson,Plymouth, UK) at a rate of 10×10⁶ cells/P10 type dish. Cells arecultured in whole RPMI 1640 medium containing GM-CSF (10 ng/ml) and IL-4(10 ng/ml) during 6 days. At day 6, cells are harvested, washed withHBSS and seeded into a 24-well plate at a density of 5×10⁵ cells/well in500 μl of whole RPMI medium and stimulated with LPS (10 μg/ml),OM-294-MP (10 μg/ml) or OM-294-DP (10 μg/ml). α TNF as well as IL-12 p70are assayed by ELISA in culture supernatants which are recovered after4, 6 and 24 hours.

Products: OM-294-MP and OM-294-DP products (1 mg/ml stock solution insterile water) are incubated at 37° C. during 20 min. and kept undervigorous stirring during 3 minutes then diluted to 100 mg/ml and used ata final concentration of 10 μg/ml in RPMI 1640 culture medium.

Reference Product: E. coli lipopolysaccharides (LPS, DIFCO, Detroit,Mich., USA), 5 mg/ml stock solution in PBS, intermediate solution inculture medium: 100 μg/ml, used at a final concentration of 10 μg/ml.

Assay of α TNF and IL-12 p70: α TNF KHC3012 kit from Biosource, batch #PP003-J061703 (Biosource International, Camarillo, Calif., USA) ELISAwas run according to the supplier's instruction manual. IL-12 p70 isassayed in culture supernatants by ELISA using the human IL-12 kit (No.D1200, batch 990 6232, R&D Systems, Minneapolis, Minn., USA).

Results

α TNF

OM-294-MP stimulates α TNF production by DC-6 cells in a way similar toLPS both with respect to time-course production rate and α TNFconcentration. (FIG. 10)). An α TNF peak is noted for both productsbetween 6 hr. and 24 hr.

OM-294-DP has only a minor stimulating effect on α TNF production byDC-6 cells.

IL-12 p70

Generally speaking, IL-12 is induced in presence of γ IFN (LPS+γ INF,OM-294-MP+γ IFN) in monocytes ((FIG. (12)) and DC-6 cells ((FIG. 11)).Cytokine production onset is earlier in DC than in monocytes.

8. Evaluation of OM-294-MP and OM-294-DP Adjuvant Properties in a MurineImmunization Model with a Synthetic Peptide (Pb CS His₆-242-310) of theC-Terminal Region of Plasmodium berghei Circumsporozoite SurfaceProteine

Procedure

Antigen: Pb CS (HHHHHHGGMN NKNNNNDDSY IPSAEKILEFVKQIRDSITE EWSQCNVTCGSGIRVRKRKRG SNKKAEDLTL EDIDTEI) peptide, hereinafter referred to asHis₆-242-310, corresponding to 242-310 amino acid sequence of Plasmodiumberghei circumsporozoite ANKA strain surface proteine plus an N-terminalstretch of 6 histidine, 2 glycine and one methionine residues isobtained by the Merrifield and Atherton synthesis method (Athenon etal., Bioorg Chem., 8: (1979) 350-351). The polypeptide was prepared on ap-alkoxybenzylacohol resin (Wang resin) having a substitution rate of0.4 mmol/g. A 10-fold molar exess of F-moc amino acid derivatives isused with a coupling time of 30 min. The peptide is purified by sizeexclusion chromatography (Sephadex G25, Pharmacia, Sweden) then byreverse phase chromatography (W-Porex 5 C-4, 250×10 mm, Phenomenex,Torrance, Calif., U.S.A) using a 40-min. gradient, ranging from a 10-50%acetonitrile-0.1% trifluoroacetic acid mixture (v/v), at a 3 ml/min.flow rate. The amino acid composition of the peptide is determinedaccording to the method of Knecht and Chang (Anal. Chem., 58 (1986)2373-2379) and the molecular weight is checked by mass spectrometryusing a Voyager DE model apparatus (Perspective Biosystem, Framingham,Mass., USA). Antigen stock solution is prepared at a concentration of0.4 mg/ml in 0.9% NaCl/water at pH 8.0.

Adjuvants: Stock solutions of OM-294-DP and OM-294-MP are prepared at aconcentration of 1 mg/ml in 0.9% NaCl-water, incorporating 0.1%triethylamine for OM-294-MP. The positive control is comprised ofIncomplete Freund's Adjuvant (IFA from Difco, Detroit, Mich., USA) withthe negative control being a 0.9% NaCl solution.

Antigen-adjuvant mixture: One volume of antigen and one volume ofadjuvant are mixed by vortexing for 3 min.

Immunization regimen: 6-week old female BALB/c mice (6 mice per group)are immunized three times, with a subcutaneous shot in the tail endcontaining 0.1 ml of the following mixtures: adjuvant antigen number ofGroup 0.05 mg/injection 0.02 mg/injection mice 1 — Pb CS His₆-242-310 62 IFA Pb CS His₆-242-310 6 3 OM-294-MP Pb CS His₆-242-310 6 4 OM-294-DPPb CS His₆-242-310 6 5 OM-294-MP — 6 6 OM-294-DP — 6

Immunization and Sampling Schedual: Weeks 0 3 4 7 9 Immunizations ↑ ↑ ↑Antibody specific response ↑ ↑ ↑ ↑ CTL response ↑ ↑

Lymphoid Organ and Blood Sampling:

Serum sampling: Blood sampling is conducted at weeks 0, 3, 7 and 9.Blood is allowed to stand for 6 min. at 37° C., then is kept overnightat 4° C. Serum is subsequently frozen at −80° C. until time of antibodyassay.

Recovery of inguinal lymph nodes and spleen: A portion of each animalgroup is killed after 4 or 9 weeks, respectively. Inguinal lymph nodesand the spleen are surgically removed.

Determination of Anti-Pb CS His₆ 242-310 Antibody Titer:

Assay of antibodies specifically raised against Pb Cs His₆ 242-310antigen is performed by ELISA. Binding of antigen is done in 96-wellmicrotiter plates (Maxisorp F96, Nunc, DK) by conducting overnightincubation in a moist chamber at 4° C. with each well containing 0.1 mlof PBS (phosphate buffered saline) containing 0.001 mg/ml of Pb CS His₆242-310 antigen. Blocking of the microtiter plate is performed with PBScontaining 1% of bovine serum albumin (BSA, Fluka, Switzerland). Platesare washed with PBS containing 0.05% Tween 20 (Sigma, St. Louis, Mo.,USA). Serum samples collected at 0, 3, 7 and 9 Weeks are seriallydiluted with dilution buffer (PBS containing 2.5% of skimmed milk powderand 0.05% of Tween 20), then transferred into a microtiter plate andallowed to stand for 1 hr. at room temperature (RT). Plates are thenwashed with PBS, a diluted solution containing mouse polyclonalanti-immunoglobulin coupled to alkaline phosphatase (Sigma, St. Louis,Mo., USA) is then dispensed into those plates and incubated for 1 hr. atRT. Plates are washed with PBS and specific antibodies are revealed by acolor reaction by adding the alkaline phosphatase substrate,p-nitrophenylphosphate (Sigma, St. Louis, Mo., USA). Absorbance at 405nm is read with a microtiter plate reader (Dynatech 25000 ELISA reader,Ashford, Middlesex, UK), each serum sample is measured in duplicate.Results stand for the mean of all measurements relating to mice in eachgroup. The antibody titer is given by the highest dilution giving asignificantly positive response, i.e. an OD greater to background noiselevel±3 SD.

ELISPOT Assay:

Antibodies specifically directed to murine γ-interferon (O1E703B2) arebound by running an overnight incubation at 4° C. in a moist chamber,adding an antibody solution at 50 μg/ml in an ELISPOT microtiter platewith the well bottom being covered by nitrocellulose (Millipore,Moisheim, France). The blocking step is effected by adding DMEM medium(Life Technologies, Grand Island, N.Y., USA) containing 10% of foetalcalf serum (FCS, Fakola, Switzerland) and letting stand for 2 hours at37° C. Cells obtained from lymphoid organs (inguinal lymph nodes andspleen) are cultured in microtiter plates at a density of 200 000cells/well, then co-cultured during 24 hours at 37° C. with 100 000 P815cells either challenged or not with the Pb CS 245-252 short peptide.After incubation, cells are removed and following the washing step, asecond murine anti-γ IFN antibody-biotin complex (ANI, 2 μg/ml in PBSwith 1% BSA) is added and incubated for 2 hr. A streptavidin-alkalinephosphatase conjugate (Boehringer Mannheim, Mannheim, GFR) is added andincubated for 1 hr at 37° C., and thereafter 3 washings are effectedwith PBS containing 0.05% Tween 20, followed by 3 washings with PBS.Presence of anti-γ IFN immune complexes is demonstrated by addingBCIP/NBT substrate (Sigma, St. Louis, Mo., USA). This reaction isstopped by washing with tap water. Spots which are positive for δ IFNare then counted under a stereomicroscope. Specific spot count is thedifference between spots counted in presence of cells challenged withpeptide and spots counted in absence of peptide. Results are given asmean measurement values recorded for mice in each group. They areexpressed as the number of spots per million of cultured blood cells.

Results

Antibody response: Production of antibodies specifically directed to PbCS His₆-242-310, as determined by ELISA, is graphically depicted formice administered one, two and three immunization shots. The controlinvolves one single shot of antigen alone and results in a very weakantibody titer. Antibody titer achieved with one single antigen shot inadmixture with OM-294-MP or respectively OM-294-DP is almost as high asthe response observed with Incomplete Freund's Adjuvant (IFA) inadmixture with the same antigen (FIG. (13)). After two shots, OM-294-MPand OM-294-DP adjuvants are able to elicit a serological response whichis respectively greater than that of IFA (FIG. (14)). Following threeshots, OM-294-MP and OM-294-DP adjuvants are able to elicit aserological response which is greater than that of IFA (FIG. (15)).

FIG. (13) ELISA, run 3 weeks after the first immunization shot.

FIG. (14) ELISA, run 4 weeks after the second immunization shot.

FIG. (15) ELISA, run 2 weeks after the third immunization shot.

FIG. (16) Antibody titer as measured before and after one, two and threeimmunization shots.

Antibody titers of animals in each group before an immunization shot andafter one, two and three immunization shots are given as mean values(FIG. (16)).

CTL Response: Recognition of the T-cell epitope Pb CS 245-252 present inPb CS His₆-242-310 peptide previously used for immunization is welldemonstated by the ELISPOT test. T-lymphocyte response recorded inimmunized animals (inguinal lymph nodes and spleen, recovered one weekafter the second shot and respectively two weeks after the third shot)is demonstrated by the rise in positive spot count for γ interferon (γIFN). Results set forth in the FIGS. (17, 18, 19 and 20) are calculatedas mean value of measurements achieved with each dilution and for miceof each group. They are expressed in number of spots per million cellsin culture.

Both OM-294-MP and OM-294-DP adjuvants result in a very significantincrease in CTL response of lymphocytes derived from spleen and inguinallymph nodes. Spleen responses are higher than those of inguinal lymphnode responses. CTL activity induced by OM-294-MP and OM-294-DPadjuvants is clearly superior to the one induced by IFA.

9. Demonstrating Non Covalent OM-294-Antigen Complexes by CapillaryElectrophoresis

Capillary electrophoresis is used in this example to demonstrate noncovalent complex formation between OM-294-DP and Pb CS His₆-242-310peptide during formulation of the vaccine preparation.

Procedure

Analysis method: 20 mM sodium borate buffer (di-sodium tetraboratedecahydrate, Merck, N^(o) 6306) pH adjusted to 7.4 with 1N NaOH (FlukaN^(o) 72072)

Zone-divided capillary tube (ungrafted), length 30 cm, diameter 50 μm.

Detection is performed at 200 nm using a Beckman PACE MDQ modelapparatus (Beckman, Brea, Calif., USA).

Separation Conditions Time [min.] process Pressure Solvent 0.00Capillary wash 20.0 psi H₂O 3.00 Capillary wash 20.0 psi 1N NaOH 6.00Sample injection  0.5 psi Borate buffer 6.08 Separation 30.0 KV Boratebuffer

Antigens: Pb CS His₆-242-310 synthetic peptide at 1 mg/ml in H₂O

Adjuvant: OM-294-DP at 1 mg/ml in H₂O

Antigen-adjuvant mixture: 250 μg/ml+250 μg/ml

Results

Antigen-adjuvant complex formation as observed in formulating thisvaccine preparation is demonstrated on the electrophoresis diagram bythe disappearance of the adjuvant peak and a shift in the antigen peakgiving rise to a new peak which is specific of the newly formed complex(FIG. (21)).

10. Treatment of Peritoneal Carcinoma Induced by Injection of CellsDerived from PROb Syngenic Tumoral Line in BDIX Rats

This experiment is aimed at demonstrating an antitumoral effect ofOM-294-DP when administered in a series of i.v. shots to rats bearingmacroscopic tumors of a few mm in size.

Procedure

Animals: In-bred BDIX rat strain was established by H. Druckrey in 1937.A pair of rats from Fribourg Max Planck Institute (RFA) was the initialsource of this colony which has been maintained since 1971 in thelaboratory animal house facility by the single line system. According tothis system, one single pair of sister-brother subjects is selected togive rise to the descendants of the next generation. Rats used in thiswork are from the Experimental Animal Breeding Center in Iffa-Credo(Arbresle, France) which has been conducting the breeding on behalf ofthe applicant's laboratory. Rats used are males aged 3 months±1 week.

Tumor Induction by PROb Cell Injection:

PROb Cell origin: DHD/K12 cell line was initially derived from a graftof a colon carcinoma fragment induced in an in-bred BDIX rat by1,2-dimethylhydrazine treatment. This line of adherent cells wassubdivided into two sublines depending on their sensitivity to trypsintreatment, and cells which are hard to break-off were given the name ofDHD/K12-TR. DHD/K12-TR cells when injected in syngenic BDIX rats giverise to steady-developing tumors. This line has been cloned, onlyDHD/K12-TRb clone referred to as PROb was used in the presentinvestigation.

Culture conditions: Adherent PROb cells were cultured in sealed bottles(Falcon, Becton Dickinson, N.J., USA) at 37° C. in complete medium madeup of Ham's F10 medium (Bio-Whittaker, Walkersville, USA) to which 10%foetal calf serum (FCS, Anval, Betton, France) were added. Replacementof culture medium was done every 3 days. Upon reaching confluency, cellswere released or scraped off within 3 to 5 min from the support by 2 mlof EDTA/trypsin solution, following 3 rinses with 2 ml of the samesolution for 2-3 minutes; cells were resuspended into whole medium, withaddition of FCS to stop trypsin action. The absence of contamination incells of mycoplasma and bacterial origin was checked at regularintervals by DNA dyeing with Hoechst fluorochrome 33258 (Aldrich Chimie,Steinheim, GFR).

Induction of peritoneal carcinoma: PROb cells are released from theirsupport as set forth in “culture conditions” section and are counted ina trypan blue coloring agent solution as a means for assessing cellviability. Cells are suspended in Ham's F10 medium. Peritonealcarcinomas are induced by intraperitoneal injection (i.p.) of 106 viablePROb cells into a syngenic BDIX rat under ether anesthesia. Tumor cellinjection is done on Day 10. In these conditions, all rats develop aperitoneal carcinoma with production of bloody ascites and die betweenthe 6^(th) day and the 12^(th) day following cell injection.

Treatment of peritoneal carcinoma: Treatment is initiated 13 days afterinjection of tumoral cells when carcinoma masses are made up of nodulesof a few mm in diameter. Treatment is administered by 10 i.v. injectionsof OM-294-DP at a rate of 1 mg/kg of body weight and in doses of 0.6mg/ml dissolved in 0.9% NaCl solution. Injections are administered 3times a week (monday, wednesday and friday) in the penile vein. Controlgroup is treated with vehicle alone i.e. 0.9% NaCl.

Assessing treatment efficiency: At D42, 6 weeks after injecting tumorcells, rats are killed and dissected, carcinoma development is assessedthrough a blind study. Measuring carcinoma volume is not feasable,instead, it is possible to classify carcinomas into 4 different types.Five classes are defined according to the number and diameter ofnodules.

Class 0: No nodules are visible

Class 1: Nodules of 0.1 to 0.2 cm in diameter are readily counted

Class 2: Countless nodules of 0.1 to 0.5 are seen

Class 3: Peritoneal cavity is invaded by nodules, some of which measure1 cm in diameter.

Class 4: Cavity is completely invaded with tumor masses of a few cm insize.

Ascite volume and animal weight change: Ascite volume is measured byweighing animals twice. A group of control rats, to which no treatmentbut only 0.9% NaCl injections had been administered, allows normaldevelopment of carcinomas to be assessed and treatment follow-up.

Assessing treatment efficiency: Survival rate of rats in treated groupsis compared to survival rate in control groups; carcinoma and ascitevolumes of rats in treated groups are compared to measurements derivedfrom rats in the control group.

Statistical analysis: Statistical significance of immunotherapy effectis determined using Kruskal-Wallis test for classifying carcinomas.Likewise, a variance analysis test is conducted for ascite volume data,and a log rank test is performed for survival rates.

Results

Carcinomas: OM-294-DP shows a remarkable anti-tumor activity in thismodel. This activity is more specifically demonstrated by the number oftumor-free animals (class 0) and further the difference with NaClcontrol is significant (p<0.05) for tumor volume. Impact of treatmentwith OM-294-DP on ascite volume is also significant (p<0.05). TABLE (a)Carcinoma classification and ascite volume Number of rats suffering fromcarcinoma Volume ascite belonging to class Effect of in ml/rat Treatment0 1 2 3 4 product (*) range mean ± σ Effect of product (**) NaCl⁽¹⁾ 1 01 0 7 — 0-84 38 ± 29 — OM-294-DP⁽²⁾ 4 1 2 1 2 p < 0.05 0-73  8 ± 23 p <0.05(*) Kruskall-Wallis test,(**) Variance analysis.⁽¹⁾8 to 10 rats died from cancer before being killed, 1 rat at D34displayed nodules and jaundice but no accurate class determination couldbe made (cannibalism), 1 rat at D37 belonged to (class 4), 2 at D38belonged to (class 4); 1 at D39 belonged to (class 4), 2 at D40 belongedto (class 4) and 1 at D41 belonged to (class 4). One rat of class 0displayed upon dissection a subcutaneous tumor, it is likely thatinjection of cancer cells failed.⁽²⁾1 rat died at D14 at the time of the first treatment injection, thelatter did not suffer from carcinoma. One of the rats killed in class 0showed a subcutaneous tumor (injection of cancer cells failed).

Survival:

Animals are killed at D42. Survival was determined at day 42 followinginjection of tumor cells, 90% of animals treated with OM-294-DPsurvived, whereas for the untreated group only 20% of animals were stillalive.

Rat survival rate is significantly extended by OM-284-DP (p<0.001).

Weight: OM-294-DP has no significant effect on weight change incomparison to animals administered NaCl alone as demonstrated by data inTable (b). TABLE (b) Weight change in rats (mean ± standard deviation)Days NaCl OM-294-DP 0 314 ± 19 277 ± 19 13 337 ± 18 310 ± 21 20 342 ± 20304 ± 23 29 361 ± 23 317 ± 24 41 314 ± 38 327 ± 26

11. Evaluation of OM-294-DP Adjuvant Properties in a Murine ImmunizationModel Through Nasal Administration of Urease B Subunit of Heliobacterpylori

It has been shown that mice can be protected from Heliobacter pyloriinfection when immunized with urease B subunit of Heliobacter pylorieither by oral or nasal administration in presence of a cholera toxin(CT) based adjuvant. Corthésy-Teulaz I., et al., Gastroenterology, 109:(1995): 115; Michetti P. et al., Gastroenterology, 116: (1999) 804;Saldinger P. F. et al., Gastroenterology 115: (1998) 891. This anti-UreB humoral response as measured in serum of immunized mice is mainly ofthe IgG1 type (Th2 response). Adjuvant effect evaluation of OM-294-DP isconducted in BALB/c mice (n=6) immunized four times at intervals of oneweek by nasal administration of urease B subunit (UreB) of recombinantHeliobacter pylori in presence of OM-294-DP. BALB/c control mice wereimmunized with OM-294-DP adjuvant alone. Two weeks following the lastbooster, blood is sampled from each mouse to assay serum anti-UreBimmunoglobulins by ELISA (total IgG, IgG1 and IgG2a).

Procedure

Animals: BALB/c/Ola/HsD mice (Harland, Horst, Netherlands): 24 mice.

Antigen: HpUreB 1-569, expressed as recombinant protein in E. coli (M15strain, Qiagen, Hilden, GFR) according to a previously described method(Michetti et al., Gastroenterology, 107: (1994) 1002).

Adjuvant: OM-294-DP (2.2 mg/ml stock solution)

Immunization Regimen:

Four groups containing 6 mice each were formed:

Groups A: 6 BALB/c mice were immunized four times by nasaladministration of 25 μg of OM-294-DP alone (25 μl per dose) once a weekthroughout 4 consecutive weeks.

Group B: 6 BALB/c mice were immunized four times by nasal administrationof 50 μg of Ure B 1-569±25 μg OM-294-DP (25 μl per dose) once a weekthroughout 4 consecutive weeks

Two weeks after the last nasal route immunization, blood was sampled bytail puncture from each mouse in groups A & B.

Assay for Serum IgG:

Coating buffer (pH 9.6): per 1 liter, Na₂CO₃ (15 mM, 1.59 g), NaHCO₃(34.8 mM, 2.93 g), Thimerosal (0.01%); PBS-Tween Buffer pH 7.4: per 1liter, NaCl (137 mM, 8.0 g), KH₂PO₄ (1.5 mM, 0.2 g), Na₂HPO₄ (8.0 mM,1.15 g), KCl (2.7 mM, 0.2 g), Tween 20 (0.1% 1 ml); Citrate/phosphatebuffer pH 5.0: per 1 liter, citric acid (44.4 mM, 9.32 g), Na₂HPO₄ (103mM, 14.6 g); Substrate solution 10× (O-phenyldiamine=OPD) (10-foldconcentrated): (10 mg/ml in citrate buffer); Sodium azide solution: 1%,Stop solution: 0.01% sodium azide in citrate/phosphate buffer 0.1 M pH5.0

Method: An antigen solution (UreB 1-569 prepared on May 26, 1999, 0.5mg/ml stock solution) is prepared at a concentration of 5 μg/ml incoating buffer pH9.6 (500 μl of UreB solution per 50 ml of buffer). 100μl are pipetted into each well in 3 round bottom 96-well plates (0.5 μgUreB per well). The plates are incubated for 2 hours at 37° C.Supernatant fluids are discarded from the plates. Wells are blocked byadding 100 μl of PBS-0.1% Tween solution+5% milk powder per well. Platesare incubated for 30 minutes at 37° C. The blocking solution isdiscarded and wells are washed 3 times with 100 μl of PBS-Tween.Supernatant fluids are discarded. A 1:200 dilution of each mouse serumto be tested is prepared in PBS-0.1% Tween buffer (5 μl of serum in 1 mlof PBS-Tween). Sera (100 μl) are divided in duplicates into three plates(1 plate to detect total IgG, 1 plate to detect IgG1 and 1 plate todetect IgG2a). Incubation is carried out overnight at 4° C. Wells arewashed 3 times with 100 μl of PBS-Tween. A 1:500 dilution ofbiotin-coupled-anti-IgG total antibody (Amersham, Cat # RPN 1177),anti-IgG1 antibody (Amersham Cat # RPN 1180) and anti-IgG2a antibodysolutions (Pharmingen Cat #˜02012D) in PBS-Tween buffer is individuallyprepared. 100 μl of the anti-IgG total antibody solution are added toplate N^(o) 1, 100 μl of the anti-IgG1 solution to plate N^(o) 2 and 100μl of the anti-IgG2a antibody solution to plate N^(o) 3. Incubation iscarried out for 1 hour at 37° C. Wells are washed 3 times withPBS-Tween. A 1:1000 dilution of streptavidin-HRP (Dako, Cat # p0397) isprepared in PBS-Tween buffer and 100 μl of the solution are added perwell. Incubation is carried out for 1 hour at 37° C. The wells arewashed 3 times with PBS-Tween buffer. A substrate solution is preparedby diluting 10-fold the OPD solution (10×) in 0.1 M citrate/phosphatebuffer. 1 μl of H₂O₂ is added to the dilute OPD solution. 50 μl of thesubstrate solution are added to each well. Color development is allowedto occur for 10 to 20 minutes. The reaction is terminated by adding 50μl of stop buffer. Absorbance is read at 492 nm (with a standard beingread at 620 nm) using a negative control as blank.

Statistical analysis: Data stand for mean±SD (n=6). p values are derivedfrom t-Student test. Data significance level is set at p<0.05.

Results

Previously immunized mice with UreB-1-569+OM-294-DP through nasal routeadministration develop an anti-UreB humoral immunity: anti-UreB 1-569IgG1 are present in the blood.

Presence of antibodies specifically directed against UreB of Hp in mouseserum is measured by ELISA. UreB (0.5 μg/well) is dispensed into roundbottom 96-well plates together with carbonate buffer pH 9.6. Specificantibodies are detected by means of rabbit anti-IgG total antibodies,anti-IgG1 and IgG2a antibodies. Results are given as optical density(OD) reading at 492 nm. OD values 3 times greater than measured valuesin serum of naive mice are considered positive. No anti-UreB antibodiesare detected in the serum of mice immunized with OM-294-DP alone. Micewhich have been immunized with Ure+OM-294-DP also develop totalanti-UreB IgG antibodies (OD=0.274±0.130, p<0.05) and anti-UreB IgG1(OD=0.212±0.128, p<0.05), but do not develop anti-UreB IgG2a antibodies(OD=0.008+0.005, non significant).

BALB/c mice immunized with urease B subunit of Heliobacter pylori bynasal administration (UreB)+OM-294-DP develop an anti-UreB humoralresponse mainly of the IgG1 type. OM-294-DP can therefore act as anadjuvant by nasal route administration and promote development of ahumoral immunity of the Th2 type.

12. OM-294-MP and OM-294-DP in Combination with H1N1 Antigen:Determining Specific Antibodies Raised in Mice After 1 or 2 SubcutaneousAdministrations

Procedure

This study is aimed at demonstrating the adjuvant effect of OM-294-MPand OM-294-DP for influenza antigen H1N1 (262195 A/B Beijinghaemagglutinin, Solvay Duphar, Weesp, NL). To this end, 60 BALB/c mice(females, 8 weeks-old at the beginning of treatment) are divided into 6groups as follows: Antigen Adjuvants Final conc.: Final conc.: 2.5 μgper 50 μg per NaCl Injected Groups animal/injection animal/injection(0.9%) volume A: NaCl — — 150 μl 150 μl B: H1N1 H1N1 (100 μl) —  50 μl150 μl C: H1N1 + H1N1 (100 μl) OM-294-MP — 150 μl OM-294-MP (50 μl) D:H1N1 + H1N1 (100 μl) OM-294-MP 100 μl 150 μl OM-294-DP (50 μl) E:OM-294-MP — OM-284-MP 100 μl 150 μl (50 μl) F: OM-294-DP — OM-284-DP 100μl 150 μl (50 μl)

Antigen: H1N1 stock solution is prepared at a concentration of 25 μg/mlin 0.9% NaCl.

Adjuvants: Stock solutions of OM-294-DP and OM-294-MP are prepared at aconcentration of 1 mg/ml in injection water, with 0.1% triethanolaminebeing added to OM-294-MP. The negative control consists of a 0.9% NaClsolution without antigen.

Antigen-adjuvant mixture: Adjuvants are kept for 20 minutes at 37° C.before vortexing for 3 minutes. Then, the antigen and NaCl (0.9%) areadded as stated in the Table given above, and the antigen-adjuvantmixture is vortexed briefly before placing it on a rotary stirrer for 15minutes at room temperature, and finally the whole mixture is vortexedfor 3 minutes.

Immunization regimen: Injections are scheduelled on days 0 and 14.Mixtures indicated in the aforementioned Table are administered bysubcutaneous route (75 μl on the side, with a total 150 μl per animal).Blood sampling is scheduelled on days 14 and 18 (orbital punctures).

Assay for anti-H1N1 immunoglobulins: The following serumImmunolglobulins which are specifically directed against H1N1 areassayed in duplicate by ELISA: IgG1, IgG2a, and IgM. Briefly stated,microtiter plates (NUNC Immunoplate, Roskilde, DK) are incubated(overnight coating) at 4° C. with 100 μl H1N1 (0.5 μg) in bicarbonatebuffer pH 9.6). After washing with 0.5% Tween-20 (Merck Hohenbrunn, D),sera are diluted 50-, 200- and 800-fold (diluting solution: phosphatebuffered saline (PBS)+1% bovine serum albumin (BSA, Sigma, St. Louis,Mo., USA)+0.02% Tween-20)). 100 μl of each dilute serum sample are addedto the wells. Incubation lasts 45 minutes at 37° C.

After a second washing step, IgG1, IgG2a and IgM specifically directedto H1N1 are incubated for 30 minutes at 37° C. together with 100 μl ofanti-IgG1 antibodies (anti-mouse rat antibody)-peroxydase conjugate(Serotec, Oxford, UK), IgG2a-peroxydase conjugate (Pharmingen, SanDiego, Calif., USA) and IgM-biotin conjugate (Pharmingen, San Diego,Calif., USA), diluted beforehand in PBS/BSA/Tween buffer (250-, 1000-,500-fold dilutions, respectively). For IgM, after an extra washing step,a 3^(rd) incubation is required (30 min. at 37° C.) with a 1:100 dilutesolution of streptavidin-peroxydase conjugate (Dako, Glostrup, DK).

After the washing step, 100 μl of a phenylene 1,2-diamine solution (OPD,Merck, Darmstadt, GFR) are added to detect peroxydase-coupled anti-IgG1and anti-IgG2 secondary antibodies (whereas for IgM, the reagent beingused is 3′,3′,5′,5′-tetramethylbenzidine (TMB, Sigma, St. Louis, Mo.,USA). After a 20 minute incubation period at room temperature, thereaction is stopped by adding 100 μl of 2N H₂SO₄. Absorbance values areread at 490 nm with a Bio-Rad 3550 model plate reader.

Results

Results of each reading at 490 nm are given in arbitrary units (A.U.)per ml. This is achieved by comparing each sample with a standardprepared from variable dilutions of a sample pool collected from group B(animals injected with H1N1 alone) at day 28. As the term implies, thesample pool diluted 50-fold is at a concentration of 1000 A.U./ml.Individual results are then corrected for the corresponding dilutionfactor (50, 200, or 800 times). Only the mean values of each group andthe standard deviation (SD) are herein reported. TABLE a Immunoglobulinsspecifically raised against H1N1 of IgG1 subclass (Arbitrary units/ml ±SD, **p < 0.01 (Anova & (two sided) Dunnetts tests). Groups Day 14 Day28 A: NaCl 3 ± 5 0 ± 0 B: H1N1 11161 ± 5755  53950 ± 23403 C: H1N1 +OM-294-MP 34411 ± 13719 228467** ± 109123  D: H1N1 + OM-294-DP 30101 ±19061 382325** ± 201314  E: OM-294-MP 69 ± 34 59 ± 31 F: OM-294-DP 59 ±21 38 ± 25

TABLE b Immunoglobulins specifically raised against H1N1 of IgG2asubclass (Arbitrary units/ml ± SD, **p < 0.01 (Anova & (two sided)Dunnetts tests). Groups Day 14 Day 28 A: NaCl 0 ± 0 0 ± 0 B: H1N1 26883± 20779 50352 ± 30846 C: H1N1 + 179344** ± 139781  1622722** ± 986195  OM-294-MP D: H1N1 + 103630 ± 96257   681441 ± 1072710 OM-294-DP E:OM-294-MP 1619 ± 743  1767 ± 1034 F: OM-294-DP 452 ± 584 782 ± 857

TABLE c Immunoglobulins specifically raised against H1N1 of IgM subclass(Arbitrary units/ml ± SD, *p < 0.05 & **p < 0.01 (Anova & (two sided)Dunnetts tests). Groups Day 14 Day 28 A: NaCl 22102 ± 5862 21531 ± 3693B: H1N1  37787 ± 15001 57306 ± 26886 C: H1N1 + OM-294-MP 67936** ±21334  95108 ± 38669 D: H1N1 + OM-294-DP 598100* ± 18324  92920 ± 26971E: OM-294-MP 19065 ± 4069 18018 ± 1016 F: OM-294-DP 20756 ± 7160 20944 ±9065

These results indicate that OM-294-MP and OM-294-DP adjuvants are activein the model under investigation, since they both significantly increasethe titer of antibodies specifically raised against H1N1 in mice afterone or two injections (see FIGS. 22, 23, 24), irrespective of theimmunoglobulin subclass being considered (IgG1a, IgG2a and IgM).

13. OM-294-OM and OM-294-DP in Combination with Ovalbumin Antigen:Determining Specific Antibodies Raised in Mice After 1 or 2 SubctuaneousAdministrations

Procedure

This study is aimed at demonstrating the adjuvant effect of OM-294-MPand OM-294-DP with regard to ovalbumin antigen (Fluka Chemie, Buchs,Switzerland). To this end, 50 BALB/c mice (females, aged 8 weeks at thebeginning of treatment) were divided into 5 groups as follows: AntigenAdjuvants Final conc.: Final conc.: 50 μg per 50 μg per Injected Groupsanimal/injection animal/injection NaCl volume A: NaCl — — 150 μl 150 μlB: Ova Ova (100 μl) —  50 μl 150 μl C: Ova + Ova (100 μl) OM-294-MP (50μl) — 150 μl OM-294-MP D: Ova + Ova (100 μl) OM-294-DP (50 μl) — 150 μlOM-294-DP E: OM-294- — OM-294-MP (50 μl) 100 μl 150 μl MP

Antigens: An ovalbumin stock solution is prepared at a concentration of0.5 mg/ml in 0.9% NaCl.

Adjuvants: Stock solutions of OM-294-DP and OM-294-MP are prepared at aconcentration of 1 mg/ml in water for injection, with 0.1%triethanolamine being added for OM-294-MP. The negative control consistsof a 0.9% NaCl solution without antigen.

Antigen-Adjuvant mixture: Adjuvants are allowed to stand for 20 minutesat 37° C. before vortex treatment for 3 minutes. Then the antigen and(0.9%) NaCl are added as specified in the above Table, and theantigen/adjuvant mixture is vortexed briefly before placing it on arotary stirrer for 15 minutes at room temperature, then the wholemixture is vortexed for 3 minutes.

Immunization regimen: Injections are scheduelled on days 0 and 14. Themixtures stated in the preceeding table are administered by subcutaeousroute (75 μl on the side, 150 μl in total per animal). Blood sampling isscheduelled on days 14 and 28 (orbital puncture).

Assay for anti-ovalbumin immunoglobulins: The following serumImmunolglobulins which are specifically directed against ovalbumin areassayed in duplicate by ELISA: IgG1, IgG2a, and IgM. Briefly stated,microtiter plates (NUNC Immunoplate, Roskilde, DK) are incubated(overnight coating) at 4° C. together with 100 μl ovalbumin (0.5 μg) inbicarbonate buffer pH 9.6). After washing with 0.5% Tween-20 (MerckHohenbrunn, D), sera are diluted 50-, 200- and 800-fold (dilutingsolution: phosphate buffered saline (PBS)+1% bovine serum albumin (BSA,Sigma, St. Louis, Mo., USA)+0.02% Tween-20)). 100 μl of each diluteserum sample are added to the wells. Incubation lasts 45 minutes at 37°C.

After a second washing step, IgG1, IgG2a and IgM specifically directedto ovalbumin are incubated for 30 minutes at 37° C. together with 100 μlof anti-IgG1 antibodies (anti-mouse rat antibody)-peroxydase conjugate(Serotec, Oxford, UK), IgG2a-peroxydase conjugate (Pharmingen, SanDiego, Calif., USA) and IgM-biotin conjugate (Pharmingen, San Diego,Calif., USA), diluted beforehand in PBS/BSA/Tween buffer (250-, 1000-,500-fold dilutions, respectively). For IgM, after an extra washing step,a 3^(rd) incubation step is required (30 min. at 37° C.) with a 1:100dilute solution of streptavidin-peroxydase conjugate (Dako, Glostrup,DK).

After the washing step, 100 μl of a phenylene 1,2-diamine solution (OPD,Merck, Darmstadt, GFR) are added to detect peroxydase-coupled anti-IgG1and anti-IgG2 secondary antibodies (whereas for IgM, the reagent beingused is 3′,3′,5′,5′-tetramethylbenzidine (TMB, Sigma, St. Louis, Mo.,USA). After a 20 minute incubation period at room temperature, thereaction is stopped by adding 100 μl of 2N H₂SO₄. Absorbance values areread at 490 nm with a Bio-Rad 3550 model plate reader

Results

Results of each reading at 490 nm are given in arbitrary units (A.U.)per ml. This is achieved by comparing each sample with a standardprepared from different dilutions of a sample pool collected from groupB at day 28 (animals injected with ovalbumin alone). As clearlyunderstood by this term, the sample pool diluted 50-fold is at aconcentration of 1000 A.U./ml. Individual results are then corrected forthe corresponding dilution factor (50, 200, or 800 times). Only the meanvalues of each group and the standard deviation (SD) are hereinreported. TABLE a Immunoglobulins specifically raised against ovalbuminof IgG1 subclass (Arbitrary units/ml ± SD, **p < 0.01 (Anova & (twosided) Dunnetts tests). Groups Day 14 Day 28 A: NaCl 6 ± 6 16 ± 12 B:ova 728 ± 589 47743 ± 46294 C: ova + OM-294-MP 4361** ± 2513  284121** ±164822  D: ova + OM-294-DP 3240** ± 1794  277025** ± 173737  E:OM-294-MP 19 ± 8  40 ± 69

TABLE b Immunoglobulins specifically raised against ovalbumin of IgG2asubclass (Arbitrary units/ml ± SD, *p < 0.05 **p < 0.01 (Anova & (twosided) Dunnetts tests). Groups Day 14 Day 28 A: NaCl 2996 ± 898  5414 ±1554 B: ova 5201 ± 1880  73162 ± 107954 C: ova + OM-294-MP 9524 ± 6809625663* ± 681232  D: ova + OM-294-DP 18108** ± 14958  601434* ± 624166 E: OM-294-MP 11253 ± 12169 4192 ± 2104

TABLE c Immunoglobulins specifically raised against ovalbumin of IgMsubclass (Arbitrary units/ml ± SD) Groups Day 14 Day 28 A: NaCl 14009 ±6158  12288 ± 7136  B: ova 19423 ± 13778 47998 ± 34035 C: ova +OM-294-MP 21652 ± 9524  38240 ± 8822  D: ova + OM-294-DP 25762 ± 10975 74399 ± 119781 E: OM-294-MP 19742 ± 5667  9827 ± 2021

These results indicate that OM-294-MP and OM-294-DP adjuvants are activein the present model under investigation, since they both significantlyincrease (with respect to IgG1 and IgG2a subclasses) the titer ofantibodies specifically raised against ovalbumin in mice after one ortwo injections (see FIGS. 25, 26, 27).

14. OM-294-MP and OM-294-DP in Combination with TT Antigen, (TetanosToxoid): Determining Specific Antibodies Raised in Mice After 1 or 2Subcutaneous Administrations

Procedure

This study is aimed at demonstrating the adjuvant effect of OM-294-MPand OM-294-DP for TT antigen (Massachussetts Biologic Laboratoires, MA,USA). To this end, 40 BALB/c mice (females, 8 weeks-old at the beginningof treatment) are divided into 4 groups as follows: Antigen AdjuvantsFinal conc.: Final conc.: 50 μg per 50 μg per NaCl Injected Groupsanimal/injection animal/injection (0.9%) volume A: NaCl — — 150 μl 150μl B: TT TT (100 μl) —  50 μl 150 μl C: TT + TT (100 μl) OM-294-MP (50μl) — 150 μl OM-294-MP D: TT + TT (100 μl) OM-294-DP (50 μl) — 150 μlOM-294-MP

Antigen: A TT stock solution is prepared at a concentration of 0.2 mg/mlin 0.9% NaCl.

Adjuvants: Stock solutions of OM-294-DP and OM-294-MP are prepared at aconcentration of 1 mg/ml in water for injection, with 0.1%triethanolamine being added for OM-294-MP. The negative control consistsof a 0.9% NaCl solution without antigen.

Antigen-Adjuvant mixture: Adjuvants are allowed to stand for 20 minutesat 37° C. before vortex treatment for 3 minutes. Then the antigen and(0.9%) NaCl are added as specified in the above Table, and theantigen/adjuvant mixture is vortexed briefly before placing it on arotary stirrer for 15 minutes at room temperature, then the wholemixture is vortexed for 3 minutes.

Immunization regimen: Injections are scheduelled on days 0 and 14. Themixtures stated in the preceeding table are administered by subcutaeousroute (75 μl on the side, 150 μl in total per animal). Blood sampling isscheduelled on days 14 and 28 (orbital puncture).

Assay for anti-TT immunoglobulins: The following serum Immunolglobulinswhich are specifically directed against TT are assayed in duplicate byELISA: IgG1, IgG2a, and IgM. Microtiter plates (NUNC Immunoplate,Roskilde, DK) are incubated (overnight coating) at 4° C. together with100 μl TT (0.5 μg) in bicarbonate buffer (pH 9.6). After washing with0:5% Tween-20 (Merck Hohenbrunn, D), sera are diluted 50-, 200- and800-fold (diluting solution: phosphate buffered saline (PBS)+1% bovineserum albumin (BSA, Sigma, St. Louis, Mo., USA)+0.02% Tween-20)). 100 μlof each dilute serum sample are added to the wells. Incubation lasts 45minutes at 37° C.

After a second washing step, IgG1, IgG2a and IgM specifically directedto ovalbumin are incubated for 30 minutes at 37° C. together with 100 μlof anti-IgG1 antibodies-peroxydase conjugate (Serotec, Oxford, UK),IgG2a-peroxydase conjugate (Pharmingen, San Diego, Calif., USA) andIgM-biotin conjugate (Pharmingen, San Diego, Calif., USA), dilutedbeforehand in PBS/BSA/Tween buffer (250-, 1000-, 500-fold dilutions,respectively). For IgM, after an extra washing step, a 3^(rd) incubationstep is required (30 min. at 37° C.) with a 1:100 dilute solution ofstreptavidin-peroxydase conjugate (Dako, Glostrup, DK).

After the washing step, 100 μl of a phenylene 1,2-diamine solution (OPD,Merck, Darmstadt, GFR) are added to detect peroxydase-coupled anti-IgG1and anti-IgG2 secondary antibodies (whereas for IgM, the reagent beingused is 3′,3′,5′,5′-tetramethylbenzidine (TMB, Sigma, St. Louis, Mo.,USA). After a 20 minute incubation period at room temperature (40 min.for TMB), the reaction is stopped by adding 100 μl of 2N H₂SO₄.Absorbance values are read at 490 nm with a Bio-Rad 3550 model platereader

Results

Results of each reading at 490 nm when measuring IgG1 and IgG2a aregiven in arbitrary units (A.U.) per ml. This is achieved by comparingeach sample with a standard prepared from different dilutions of asample pool collected from the group B at day-28 (animals injected withTT alone). As this term implies, the sample pool diluted 50-fold is at aconcentration of 1000 A.U./ml. Individual results are then corrected forthe corresponding dilution factor (50, 200, or 800 fold). Only the meanvalues of each group and the standard deviation (SD) are hereinreported.

Regarding the assay of IgM specific for TT, since the “background noise”of the assay was too high, no clear cut difference could be foundbetween group B (TT alone) and groups C and D (TT with adjuvant) bymeasuring specific IgM as previously done for IgG1 and IgG2a. Instead,specific IgM titer could be determined, using successive dilutions ofeach sample (rather than A.U. described before) and the result wasreported, for each sample, as the highest dilution which gives anabsorbance reading greater than mean absorbance±3 SD for group A (NaCl).The titer thus obtained indicates the number of times a serum sample canbe diluted before the absorbance thereof could no longer bedistinguished from background noise level. This final dilution is theresult reported in Table (c) for IgM given hereinafter. TABLE (a)Immunoglobulins specifically raised against TT of IgG1 subclass(Arbitrary units/ml ± SD, **p < 0.01 (Anova & (two sided) Dunnettstests)). Groups Day 14 Day 28 A: NaCl 1 ± 2 0 ± 1 B: TT 2871 ± 163334367 ± 15018 C: TT + OM-294-MP 8502** ± 2020  78506** ± 21660  D: TT +OM-294-DP 11620** ± 2348   136463** ± 41025  

TABLE (b) Immunoglobulins specifically raised against TT of IgG2asubclass (Arbitrary units/ml ± SD, **p < 0.01 (Anova & (two sided)Dunnetts tests). Groups Day 14 Day 28 A: NaCl 351 ± 506  536 ± 1046 B:TT 2547 ± 2539 61387 ± 82269 C: TT + OM-294-MP 8869 ± 6979 65881 ± 46635D: TT + OM-294-DP 21969** ± 25067  148365 ± 134196

TABLE (c) Titer of immunoglobulins specifically directed against TT ofIgM subclass (dilutions for each sample giving a signal at 490 nmgreater than the mean ± 3 SD for a sample dilution In group A). GroupsDay 14 Day 28 A: NaCl standard standard B: TT 9 animals <25 10 animals<25 1 animal <50 C: TT + OM-294-MP 9 animals <25  7 animals <25 1animal >1600  1 animal <100  2 animals >1600 D: TT + OM-294-DP 7 animals<25 10 animals <25 1 animal <200 2 animals >1600

These results indicate that adjuvants OM-294-MP and OM-294-DP are activein the present model under investigation, since they both oftensignificantly increase the titer of IgG1 and IgG2a antibodiesspecifically raised against TT in mice after one or two injections (seeFIGS. 28, 29). By contrast, only a few animals produced IgM specific toTT.

15. Evaluation of the Adjuvant Properties of OM-294-MP in a CBA MouseImmunization Model by Subcutaneous Administration of Leishmania gp63Antigen.

Procedure

CBA mice are administered in the tail two subcutaneous injections ofgp63 at a dose of 2 μg at intervals of 8 days. OM-294-MP adjuvant ismixed with both doses of antigen, BCG is mixed only with the first dose.Each mouse is administered 2×50 μg of OM-294-MP or 200 μg of BCG. Acontrol group is injected with antigen alone (without adjuvant). Tendays after the second injection, the inguinal and periaortic lymph nodecells (groups of 3 mice each) are cultured and the proliferationresponse to the purified gp63 antigen is assayed by measuring (³H-TdR)thymidine take-up. In vitro cytokine production in terms of γ IFN andIL-4 by secreted by lymph node lymphocytes rechallenged in vitro withthe gp63 antigen is also determined by ELISA (MIF100 IFN and M4000 IL-4kits, R&D Systems, Europe Ltd., Abingdon, UK) on each supernatant sampleof lymph node lymphocyte culture prior to the addition of ³H-TdR.

Values reported in the tables for ³H-TdR take-up, stand for thearithmetic mean±standard deviation (triplicates) expressed in cpm and,values relating to cytokines in the supernatant fluid correspond to thearithmetic mean±standard deviation (triplicates) expressed in pg per ml.

Antigen: A gp63 stock solution is prepared at a concentration of 40μg/ml in 0.9% NaCl.

Adjuvants: The stock solution of OM-294-MP is prepared at aconcentration of 1 mg/ml in water for injection, with addition of 0.1%triethanolamine. The negative control consists of a PBS solution withoutantigen.

Antigen-Adjuvant mixture: Adjuvants are allowed to stand for 10 minutesat 37° C. before vortex treatment for 3 minutes. Then, the antigen (1volume) and the adjuvant (1 volume) are mixed and vortexed brieflybefore incubation for 20 minutes at 37° C., and finally the wholemixture is vortexed for 3 minutes.

Results

In mice immunized with gp63 antigen, OM-294-MP adjuvant induces a betterlymphocyte proliferation response (table (a) and FIG. 30 (a)) than BCG.In fact, with respect to cultures from animals that have been immunizedwithout adjuvant, the increase in proliferation rate ranges from 3.1 to6 folds for OM-294-MP product while it does not exceed 3.5 folds for BCG(2.6-3.5).

In those lymphocyte cultures, cytokine production is measured in thesupernatant fluid (Table (b) and FIG. (30 (b)). It is noted that antigengp63 induces γ IFN secretion in quantities equivalent to mice treated byOM-294-MP or BCG as adjuvant. By contrast, OM-294-MP adjuvant seems tofavor secretion of substantial quantities of IL-4 by (anti-gp63) immunelymphocytes while BCG treated murine lymphocytes secrete minor or evenundetectable quatities of said cytokine. TABLE (a) Effect of OM-294-MPadjuvant on immune response against gp63 antigen, as measured by murineT lymphocyte proliferation in response to gp63 antigen in vitro gp63 invitro without adjuvant OM-294-MP BCG (μg/ml) (cpm × 10⁻³/ml) (cpm ×10⁻³/ml) (cpm × 10⁻³/ml) 0  1.4 ± 0.4 2.8 ± 0.7 5.3 ± 1.1 0.16 18 ± 5 65± 11 47 ± 9  0.31 17 ± 5 92 ± 11 57 ± 17 0.62 16 ± 4 93 ± 13 54 ± 131.25 13 ± 2 39 ± 7  44 ± 9 

Values reported in Table (a) stand for the arithmetic meantake-up±standard deviation (triplicate cultures) TABLE (b) Effect ofOM-294-MP adjuvant administered in vivo in conjunction with gp63 antigenon in vitro production of cytokines by lymph node lymphocytes gp63 invitro without adjuvant 294-MP BCG γ IFN concentration (pg/ml) 0 <9 <9 270.3 78 135 200 0.6 38 120 105 IL-4 concentration (pg/ml) 0 <8 <8 <8 0.3<15 125 15 0.6 <8 83 <8

OM-294-MP adjuvant potentiates specific T response in vitro in CBA micewhich have been immunized with gp63 (an amphophilic antigen of theLeishmania parasite) as assayed by lymphocyte proliferation andantigen-induced γ IFN and IL-4 production.

16. Effectiveness of OM-294-MP Adjuvant During Anti-LmCPb T-PrimaryResponse in a CBA Mouse-Based Immunization Model when Administered bySubcutaneous Route with Leishmania mexicana LmCPb Antigen

Procedure

CBA mice were administered through the tail a single injection of LmCPbin a 2 μg dose either in combination or not with 50 μg of OM-294-MPadjuvant. A control group was administered one injection ofphysiological saline buffer (non immunized subjects). Eleven days later,cells of the inguinal and periaortic lymph nodes (groups of 3 mice each)were cultured and the proliferative response directed to the purifiedLmCPb antigen, to a preparation of whole amastigotes of Leishmaniamexicana and to concanvalin A (ConA) was evaluated by measuringtritiated thymidine take-up (3H-TdR). γ IFN and IL-4 cytokine productionby lymph node lymphocytes rechallenged in vitro with LmCPb antigen ofLeishmania mexicana or by amastigotes was equally determined by ELISA(MIF00 γ IFN & M4000 IL-4 kits, R&D Systems Europe Ltd, Abingdon, UK)using a sample of each culture supernatant of lymph node lymphocytesbefore addition of ³H-TdR.

Values reported in the tables stand for the mean value±standarddeviation (triplicate) expressed in, cpm for ³H-TdR take-up and thearithmetic mean±standard deviation (triplicate) expressed in pg per mlfor cytokine production in supernatant fluids.

Antigen: LmCPb stock solution is prepared at a concentration of 40 μg/mlin PBS (2×).

Adjuvants: OM-294-MP stock solution is prepared at a concentration of 1mg/ml in water for injection with addition of 0.1% triethanolamine. Thenegative control consists of a PBS solution without antigen.

Antigen-adjuvant mixture: Adjuvants are maintained for 10 minutes at 37°C. before vortexing for 3 minutes. Then, the antigen (1 volume) and theadjuvant (1 volume) are mixed and vortexed briefly before beingincubated for 20 minutes at 37° C., and finally the whole mixture isvortexed again for 3 minutes.

Results

In absence of any stimulus in the culture medium, OM-294-MP adjuvantpromotes development of lymphocytes which undergo spontaneousproliferation (Table (a) and FIG. 31 (a)) and secrete trace amounts of γIFN (Table (b) and FIG. 31(b)). This reaction is strongly potentiatedwhen purified LmCPb antigen or an extract of whole parasite is added tothe cultures. In this experiment, the adjuvant is observed to exert aclear influence on the induction of sensitized lymphocytes (anti-LmCPb)able to secrete substantial quantities of IL-4 (Table (b) and FIG. 31(b)). TABLE (a) in vitro proliferative response of lymph node cellsimmunized in vivo by LmCPb: effect of OM-294-MP adjuvant ³H-TdR take-up(cpm × 10⁻³/ml) Non Without Antigen in vitro immunized adjuvantOM-294-MP No stimulant 0.9 ± 0.3 2.2 ± 0.7 11 ± 2 LmCPb 0.6 μg/ml 0.8 ±0.4 1.7 ± 0.1 19 ± 4 LmCPb 1.7 μg/ml 0.9 ± 0.1 4.6 ± 1.6 40 ± 4 LmCPb 5μg/ml 1.3 ± 0.8 7.2 ± 0.6 80 ± 5 LmCPb 15 μg/ml 2.6 ± 0.4 16.2 ± 2.1 140 ± 10 Amastigotes 1.9 × 10⁻⁶/ml 0.8 ± 0.2 1.7 ± 0.1 44 ± 7Amastigotes 6 × 10⁻⁶/ml 1.5 ± 0.2 4.6 ± 0.6 79 ± 6 Amastigotes 17 ×10⁻⁶/ml 2.9 ± 0.6 7.2 ± 0.6 119 ± 4  Con A 5 μg/ml 123 ± 33  193 ± 17 196 ± 10

TABLE (b) In vitro secretion of cytokines by lymph node lymphocytesimmunized in vivo by LmCPb: Effect of OM-294-MP adjuvant on primaryresponse Non without In vitro stimulant immunized adjuvant OM-294-MP γIFN production(pg/ml) No stimulant <9 <9 25 LmCPb 15 μg/ml <9 46 480Amastigotes 17 × 10⁻⁵/ml <9 95 320 Con A 5 μg/ml >1800 >1800 >1800 IL-4production (pg/ml) No challenge <8 <8 <8 LmCPb 15 μg/ml <8 <8 130Amastigotes 17 × 10⁻⁵/ml <8 <8 65 Con A 5 μg/ml 92 190 360

OM-294-MP adjuvant is also very effective during the primary T-response(following a single vaccine injection). Properties of the adjuvanteffect on this response (increase of lymphocyte proliferation, inductionof cytokine production) are similar to those observed during theresponse to two vaccine injections.

17. Evaluation of OM-294-MP and OM-294-DP adjuvant properties in CBAmouse-based immunization model through subcutaneous administration ofLeishmania mexicana LmCPb antigen comparison with BCG

Procedure

CBA mice (8 mice per group) were administered in the tail 2 subcutaneousinjections of 3-5 μg of purified LmCPb at intervals of 8 days. OM-294-MPand OM-294-DP adjuvants were mixed with both doses of antigen, whereasBCG was only mixed with the first one. Each mouse received 2×50 μg of OMadjuvant or 200 μg of BCG. Eight days following the second injection,the periaortic and inguinal lymph nodes (3 mice per group) are removedand the cells are cultured in order to assay the proliferative responseto purified LmCPb antigen, or respectively, the proliferative responseto a whole preparation of Leishmania mexicana amastigotes or toConcanavalin A (Con A). The proliferative response is evaluated bymeasuring tritiated thymidine take-up (³H-TdR). γ IFN and IL-4 cytokineproduction by lymph node lymphocytes rechallenged, in vitro by the LmCPbantigen of Leishmania mexicana or by the amastigotes or by Con A isdetermined by ELISA (MIF00 γ IFN & M4000 IL-4 kits, R&D Systems EuropeLtd., Abingdon, UK) using a sample of each culture supernatant of lymphnode lymphocytes before addition of ³H-TdR.

Values reported in the tables stand for the arithmetic mean±standarddeviation expressed in % of standard regarding antibody titer, thearithmetic mean±standard deviation (triplicates) expressed in cpm for³H-TdR take-up, and the arithmetic mean±standard deviation (triplicates)expressed in pg per ml for cytokine production.

Antigen: LmCPb stock solution is prepared at a concentration of 60-100μg/ml in 0.9% NaCl.

Adjuvants: OM-294-MP and OM-294-DP stock solutions are prepared at aconcentration of 1 mg/ml in water for injection with addition of 0.1%triethanolamine for OM-294-MP. The negative control consists of a PBSsolution without antigen.

Antigen-adjuvant mixture: Adjuvants are maintained for 10 minutes at 37°C. before vortexing for 3 minutes. Then, the antigen (1 volume) and the0.9% adjuvant (1 volume) are mixed and vortexed briefly before beingincubated for 20 minutes at 37° C., and finally the whole mixture isvortexed again for 3 minutes.

Results

In mice immunized with LmCPb antigen (Tables (a) and (b), FIGS. 32 (a)and (b)), the products OM-294-MP and OM-294-DP produce a similar effectto that observed in mice which develop an immune response against gp63.Therefore, in presence of LmCPb (15 μg/ml), the extent of proliferationof cultures derived from mice which have been immunized with antigenplus. OM-294-MP and OM-294-DP adjuvants is respectively 23 and 28 timeshigher than cultures orginating from mice administered antigen alone(without adjuvant). The impact of BCG in these conditions is smaller,since the increase in proliferation is merely 11 fold. Analogous effectsare seen in cultures challenged with purified antigen or a whole extractof Leishmania parasite, and for all antigen concentrations being tested.

γ IFN production in response to LmCPb antigen tends to be a bit higherwith product OM-294-DP than with BCG (Table (b) and FIG. 32 (b)). Itshall be noted that in this experiment, lymphocytes proliferate andsecrete substantial amounts of γ IFN even though the antigen might nothave been added to the culture medium. In this case, OM-294-DP adjuvanttends to be somewhat more effective than BCG. A clear difference betweenOM-294-MP, OM-294-DP and BCG adjuvants respectively is observed asreported above regarding the development of lymphocytes able to produceIL-4. The quantity of IL-4 being produced under the influence ofOM-294-MP and OM-294-DP adjuvants is significant since it matches thequantity secreted by lymphocytes exposed to Con A, a powerful nonspecific stimulant for lymphocytes (see Table (a) and (b)). TABLE (a) Invitro proliferative response to lymph node cells derived from miceimmunized in vivo with LmCPb: effect of different adjuvants ³H-TdRtake-up (cpm × 10⁻³/ml) in vitro stimulant without adjuvant OM-294-DPOM-294-MP BCG No stimulant 1.7 ± 0.6 21.8 ± 2.2 17.1 ± 2.5 18.6 ± 4.8LmCPb 0.6 μg/ml 0.8 ± 0.3  40.9 ± 12.7 22.9 ± 2.8 19.0 ± 7.4 LmCPb 1.7μg/ml 2.4 ± 0.1  57.2 ± 10.9 34.1 ± 4.1 39.8 ± 5.7 LmCPb 5 μg/ml 2.8 ±0.6 70.2 ± 9.2 70.3 ± 6.4  44.0 ± 10.4 LmCPb 15 μg/ml 4.3 ± 0.1 100.0 ±6.5  124.2 ± 12.0 46.2 ± 0.3 Amastigotes 2 × 10⁻⁶/ml 2.4 ± 0.6 61.0 ±1.7 28.4 ± 8.3 24.4 ± 4.3 Amastigotes 6 × 10⁻⁶/ml 2.3 ± 0.7 81.5 ± 5.566.1 ± 4.5 23.6 ± 2.5 Amastigotes 17 × 10⁻⁶/ml 1.7 ± 0.4 78.2 ± 7.8 68.7± 2.3 23.4 ± 4.0 Con A 5 μg/ml 188.1 ± 21.0  135.9 ± 3.7  151.4 ± 3.7 119.7 ± 28.5Values reported in the table stand for the arithmetic mean ± standarddeviation in terms of tracer take-up (triplicate cultures)

TABLE (b) in vitro cytokine production by lymph node lymphocytes frommice immunized in vivo by LmCPb antigen: Effect of different adjuvantswithout In vitro stimulant adjuvant OM-294-DP OM-294-DP BCG γ IFNconcentration (pg/ml) No stimulant <9 460 240 280 LmCPb 15 μg/ml 44 520360 460 Amastigotes 14 >600 >600 480 17 × 10⁻⁶/ml Con A 5 μg/ml 12001200 1900 >3000 IL-4 concentration (pg/ml) No stimulant <15 <8 <8 <8LmCPb 15 μg/ml <15 110 88 36 Amastigotes <8 130 110 29 17 × 10⁻⁶/ml ConA 5 μg/ml 40 230 85 105

OM-294-MP and OM-294-DP adjuvants potentiate efficiently the immuneresponse against a soluble antigen of Leishmania, LmCPb protease. Thisis reflected in vitro by an increase in proliferative response followedby induction of γ IFN and IL-4 production in significant amounts.

EXAMPLE VI

Aqueous Solution for Injection Compound of example III 1 g Polysorbate80 0.2 g Sodium chloride 9 g Distilled water for injection q.s. 1000 ml

The solution was adjusted to pH 7.5 with 0.1 M HCl and then wassterilized by membrane filtration on a 0.22 μm Steritop Express 1000membrane (PES membrane, 90 mM, SCGP T10 RE, Millipore Corporation,Bedford, Mass., USA). The sterile solution was divided into sterileampuls of 1 ml.

Lyophilized Product Compound of example IV 2 g Polysorbate 80 0.2 gSodium chloride 9 g Mannitol 10 g Ascorbic acid 0.1 g Distilled waterfor injection q.s. 1000 ml

The solution was adjusted to pH 7.4 with 0.1M HCl then sterilized bymembrane filtration through a 0.22 μl Steritop Express 1000 membrane(PES membrane, 90 mm, SCGP T10 RE, Millipore Corporation, Bedford,Mass., USA). The sterile solution was divided into sterile multidosevials by dispensing 1 ml per vial, and then freeze-dried.

1-19. (canceled)
 20. A diaminoalcohol of the formula:

wherein R₂ designates an acyl of a saturated or unsaturated carboxylicacid of 2 to 24 carbon atoms, which is unsubstituted or bears one memberselected from the group consisting of hydroxyl, alkyl, alkoxy of 1 to 24carbon atoms and alkyl thio of 1 to 24 carbon atoms and p and q areintegers from 1 to
 10. 21. A ω-hydroxy, ω-amino or ω-thio amino acidcompound of the formula:

wherein R₁ is an acyl of a saturated carboxylic acid of 2 to 24 carbonatoms, which is unsubstituted or substituted with at least one member ofthe group consisting of hydroxyl, alkyl, alkoxy of 1 to 24 carbon atomsand alkyl thio of 1 to 24 carbon atoms m is an integer ranging from 1 to10, n is an integer from 0 to 10, and X is an acid group which isoptionally in an ester form selected from the group consisting of:carboxy [(C₁₋₅)alkyl] CH—[(CH₂)_(m1)COOH][(CH₂)_(n1)COOH] with m₁=0 to 5and n₁=0 to 5 phosphono [(C₁₋₅)alkyl]dihydroxyphosphonyloxy[(C₁₋₅)alkyl] dimethoxyphosphonyl phosphonohydroxysulfonyl hydroxysulfonyl [(C₁₋₅)alkyl] and hydroxysulfonyloxy[(C₁₋₅)alkyl]
 22. An ω-hydroxy, ω-amino acid compound of the formula:

wherein R₁ is an acyl of a saturated or unsaturated carboxylic acidhaving from 2 to 24 carbon atoms, which is unsubstituted or substitutedwith at least one member selected from the group consisting of hydroxyl,alkyl and alkoxy of 1 to 24 carbon atoms, amino, acyloxy of an organiccarboxylic acid of 1 to 24 carbon atoms. m is an integer ranging from 1to 10, n is an integer ranging from 0 to 10, and X is dialkyloxy- ordiaryloxy-phosphoryl of the formula:

wherein R is a group which readily undergoes clevage by hydrogenolysis.